Method for treating a GD2 positive cancer

ABSTRACT

Preparations and methods for treating a GD2 positive cancer by administering a preparation comprising an anti-GD2 antibody to a patient, wherein the patient is not concomitantly treated with Interleukin-2 (IL-2), and wherein one or more treatment periods with the antibody may be preceded, accompanied, and/or followed by one or more treatment periods with a retinoid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/086,696 filed 21 Nov. 2013, abandoned, which is acontinuation-in-part of International Application No. PCT/EP2012/064970filed 31 Jul. 2012, which claims priority to International ApplicationNo. PCT/EP2012/061618 filed 18 Jun. 2012. The entire contents of each ofthe above-referenced disclosures is specifically incorporated byreference herein without disclaimer.

FIELD OF THE INVENTION

In a first aspect, the present invention relates to a method fortreating a GD2 positive cancer by administering a preparation comprisingan anti-GD2 antibody to a patient as a continuous intravenous infusionover 24 hours per day.

In a second aspect, the present invention relates to preparations andmethods for treating a GD2 positive cancer by administering apreparation comprising an anti-GD2 antibody to a patient, wherein thepatient is not concomitantly treated with Interleukin-2 (IL-2), andwherein one or more treatment periods with the antibody is/are preceded,accompanied, and/or followed by one or more treatment periods with aretinoid. In particular, the invention relates to preparations andmethods for treating a GD2 positive cancer by administering apreparation comprising an anti-GD2 antibody to a patient, wherein thepatient is not concomitantly treated with Interleukin-2 (IL-2),Granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or one ormore other cytokines. Furthermore, the invention relates to preparationsand methods for the treatment of a GD2 positive cancer in a patient,wherein a preparation comprising an anti-GD2 antibody is administered tothe patient as a continuous infusion for one or more days and for two ormore treatment cycles, without concomitantly administering IL-2. Thepresent invention further relates to preparations and methods for thetreatment of a GD2 positive cancer in a patient, wherein a preparationcomprising an anti-GD2 antibody is administered to the patient as acontinuous infusion without concomitantly administering IL-2, andwherein the anti-GD2 antibody is not a 14G2a antibody.

BACKGROUND OF THE INVENTION

Neuroblastoma, after brain cancer, is the most frequent solid cancer inchildren under five years of age. In high-risk neuroblastoma, more thanhalf of the patients receiving standard therapy have a relapse andultimately die from the disease. 90% of cases occur between ages zero tosix. The worldwide incidence in industrialized countries is around 2000cases per year.

Monoclonal antibodies against specific antigens are increasingly beingused in oncology. The entirely different mode of action compared tocytotoxic therapies have made them a valuable asset as is shown byforerunners like trastuzumab, cetuximab, bevacizumab, rituximab andothers. The disialoganglioside GD2 is a glycosphingolipid expressedprimarily on the cell surface. GD2 expression in normal tissues is rareand primarily restricted to the central nervous system (CNS), peripheralnerves and melanocytes. In cancerous cells, GD2 is uniformly expressedin neuroblastomas and most melanomas and to a variable degree in boneand soft-tissue sarcomas, small cell lung cancer, renal cell carcinoma,and brain tumors (Navid et al., Curr Cancer Drug Targets 2010;10:200-209). Because of the relatively tumor-selective expressioncombined with its presence on the cell surface, GD2 represents apromising target for antibody-based cancer immunotherapy.

Accordingly, several anti-GD2 antibodies are subject to preclinical orclinical investigation in neuroblastoma, melanoma and other GD2-relatedcancers.

APN311 is a formulation of the chimeric monoclonal anti-GD2 antibodych14.18 recombinantly produced in Chinese hamster ovary (CHO) cells,which is the standard mammalian cell line for production of commerciallyavailable antibodies. In a Phase I clinical study in relapsed/refractoryneuroblastoma patients remissions were achieved with this antibody assingle agent. A Phase III trial comprising treatment with APN311 wasinitiated in 2006 by the International Society of Paediatric OncologyEuropean Neuroblastoma (SIOPEN) and is presently investigating theeffects on event-free and overall survival related to treatment withAPN311 together with isotretinoin, i.e. cis-retinoic acid (cis-RA), withor without s.c. IL-2. In a comparable US study using a treatment packageof 4 drugs, namely a related antibody produced in SP2/0 murine hybridomacells together with i.v. Interleukin-2 (IL-2 or IL2),Granulocyte-macrophage colony-stimulating factor (GM-CSF) andisotretinoin, interesting survival improvement was seen in children withneuroblastoma in complete remission following initial therapies and noevidence of disease.

APN301 is a formulation of an immunocytokine comprising a humanizedanti-GD2 antibody (hu14.18) and IL-2 as a fusion protein. The antibodyportion specifically binds to the GD2 antigen that is strongly expressedon neuroblastoma and several other cancers. IL-2 is a cytokine thatrecruits multiple immune effector cell types. In neuroblastoma patients,APN301 is designed to localize GD2-positive tumor cells via the antibodycomponent. The fused IL-2 then stimulates the patient's immune systemagainst the tumor by activation of both, NK and T cells, whereas the Fcportion of the antibody is designed to trigger tumor cell killing byantibody-dependent cellular cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC). The immunocytokine has shown activity in a Phase IIclinical study in children with relapsed/refractory neuroblastoma(Shusterman et al.; JCO 2010 28 (33):4969-75.) and was also tested in aPhase I/II study in late stage malignant melanoma, showing immuneactivation.

Other anti-GD2 antibodies in research or development are, for example,the monoclonal antibody 3F8 (murine in phase II, as well as humanized inphase I), and 8B6 (specific to O-acetylated GD2, preclinical).Furthermore, anti-idiotypic antibodies such as e.g. 4B5, 1A7, and A1G4have been under investigation as potential tumor vaccines, however,their development seems to be abandoned. WO 2008/049643 also describesanti-idiotypic antibodies, which mimic GD2 epitopes, i.e. GD2 mimotopes.

Another version of the 14.18 anti-GD2 antibody is hu14.18K322A asdescribed in WO2005/070967, which has a point mutation in the Fc regionin order to reduce CDC, but maintain ADCC, e.g. by expression in a cellline suitable for enhancing ADCC, such as YB2/0. The reduction in CDC isconsidered to result in reduced pain associated with the antibodytreatment.

Anti-tumor activity of antibodies generally occurs via either complementdependent cytotoxicity (CDC or complement fixation) or through antibodydependent cell-mediated cytotoxicity (ADCC). These two activities areknown in the art as “effector functions” and are mediated by antibodies,particularly of the IgG class. All of the IgG subclasses except IgG4(IgG1, IgG2, IgG3) mediate ADCC and complement fixation to some extent,with IgG1 and IgG3 being most potent for both activities. ADCC isbelieved to occur when Fc receptors on natural killer (NK) cells and/orother Fc receptor bearing immune cells (effector cells) bind to the Fcregion of antibodies bound to antigen on a cell's surface. Fc receptorbinding signals the effector cell to kill the target cell. CDC isbelieved to occur by multiple mechanisms; one mechanism is initiatedwhen an antibody binds to an antigen on a cell's surface. Once theantigen-antibody complex is formed, the C1q molecule is believed to bindthe antigen-antibody complex. C1q then cleaves itself to initiate acascade of enzymatic activation and cleavage of other complementproteins, which then bind the target cell surface and facilitate itsdeath through, for example, cell lysis and/or ingestion by macrophages.

However, CDC is considered to cause the side effect of pain, especiallyfor anti-GD2 antibodies. As described in WO2005/070967, neurons may beparticularly sensitive to complement fixation because this processinvolves the creation of channels in a cell membrane, allowing anuncontrolled ion flux. In pain-sensing neurons, even a small amount ofcomplement fixation may be significant to generate action potentials.Thus, any amount of CDC resulting from anti-GD2 antibody binding onneurons will result in pain. Accordingly, the prior art teaches that itis advantageous to reduce complement fixation so as to reduce the levelof side effects in a patient and that the antitumor activity of anti-GD2antibodies results primarily from ADCC, and not substantially fromcomplement fixation (see e.g. WO2005/070967).

In contrast, a key aspect of the invention is that the cytolysiscapacity of an anti-GD2 antibody determined by a CDC assay or a wholeblood test (WBT) is essential for the anti-tumor effect of the anti-GD2antibody. Such a WBT assay in contrast to CDC or ADCC assays measuresthe lytic potential of a heparinized whole blood sample. Thus, it doesnot only focus on one single effector mechanism but measures acombination of ADCC and CDC (and any other components and/or mechanismspresent in the heparinized whole blood sample which might also berelevant to the lytic capacity against tumor cells) in a physiologicalsetting. Accordingly, with the methods of the present invention it ispossible to reduce the dose of the antibody to the minimal dose requiredfor target cell lysis as determined by a CDC assay or a WBT.Furthermore, the methods of the invention allow to individuallydetermine the effective antibody dose and thus, take into account theindividual differences in anti-tumor responses of the patients. Anotherkey aspect of the invention is that it is possible to reduce and managethe side effect of pain by determining the threshold dose of theanti-GD2 antibody to be administered to induce CDC and/or whole bloodcytolytic activity. Another key finding of the invention is that theside effect of pain can be substantially reduced by administering theanti-GD2 antibody as a continuous infusion until the predeterminedoverall patient dose has been administered. Accordingly, with themethods according to the invention it is possible to substantiallyreduce the analgesic administration, especially the administration ofstrong analgesics such as morphine, during the antibody treatment, andthus, also substantially reduce the side effects of such analgesicadministration.

As stated above, it is believed that antibody-dependent cellularcytotoxicity (ADCC) plays an important role in immunotherapy.Unfortunately, ADCC is often depressed in cancer patients. Cytokines areconsidered to augment ADCC by direct activation of immune cells or byenhancement of tumor-associated antigens (TAA) on tumor cells. Forexample, Aldesleukin (IL-2) causes activation of natural killer (NK)cells, generation of lymphokine-activated killer (LAK) cells, andaugments ADCC. Aldesleukin (IL-2) has been effective at inducingmeasurable antitumor responses in patients with renal cell carcinoma andmelanoma. Furthermore, GM-CSF has been shown both in vitro and in vivoto enhance antitumor immunity through direct activation of monocytes,macrophages, dendritic cells, and antibody-dependent cellularcytotoxicity (ADCC), and indirect T cell activation via TNF, interferonand interleukin 1 (IL-1). GM-CSF is considered to enhance functions ofcells critical for immune activation against tumor cells, alone or withother cytokines or monoclonal antibodies.

Thus, in current clinical trials investigating anti-GD2 antibodies, inparticular ch14.18, the antibody treatment is combined with cytokinetreatment (and retinoid treatment), especially with IL-2 and/or GM-CSF.Accordingly, the prior art teaches that it is advantageous to administercytokines to GD-2 positive cancer patients, in particular in combinationwith anti-GD2 antibody treatment.

In contrast, another key aspect of the invention is that such patientscan be treated with an anti-GD2 antibody without IL-2, especiallywithout any cytokine treatment.

The treatment with one or more cytokines in combination with theantibody may have severe side effects, such as e.g. fever, allergicreactions, hypotension, capillary leak syndrome etc., which may evenlead to death. The accompanying cytokine treatment even potentiatesadverse events of the antibody treatment, e.g. pain, since there is asynergy in adverse effects of both drugs. However, with the preparationsand methods of the present invention, it is possible to completely omitany cytokine(s), especially IL-2. Thus, the present invention results insubstantially reduced adverse effects of the treatment with theantibody.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a method fortreating a GD2 positive cancer by administering a preparation comprisingan anti-GD2 antibody to a patient as a continuous intravenous infusionover 24 hours per day. Said preparation comprising an anti-GD2 antibodymay be administered by using a mini-pump, and may be administered for atreatment period until the predetermined overall patient dose has beenadministered.

Furthermore, the present invention relates to a method for treating aGD2 positive cancer by administering a preparation comprising ananti-GD2 antibody to a patient, wherein the preparation is administeredin a dose sufficient to induce tumor cell lysis (cytolysis thresholddose), and wherein said cytolysis threshold dose is administered for atreatment period until the predetermined overall patient dose has beenadministered.

Moreover, the invention provides an anti-GD2 antibody for use in saidtreatment. In addition, the invention provides the use of an anti-GD2antibody in the preparation of a medicament for said treatment.

In a second aspect, the present invention relates to preparationscomprising an anti-GD2 antibody for use in the treatment of a GD2positive cancer in a patient, wherein the preparation comprising ananti-GD2 antibody is administered to the patient without concomitantlyadministering IL-2, and wherein one or more treatment periods with theantibody is/are preceded, accompanied, and/or followed by one or moretreatment periods with a retinoid.

Furthermore, the present invention relates to preparations comprising ananti-GD2 antibody for use in the treatment of a GD2 positive cancer in apatient, wherein the preparation comprising an anti-GD2 antibody isadministered to the patient as a continuous infusion for one or moredays and for two or more treatment cycles, without concomitantlyadministering IL-2.

Also, the present invention relates to preparations comprising ananti-GD2 antibody for use in the treatment of a GD2 positive cancer in apatient, wherein the preparation comprising an anti-GD2 antibody isadministered to the patient as a continuous infusion withoutconcomitantly administering IL-2, and wherein the anti-GD2 antibody isnot a 14G2a antibody.

Moreover, the invention relates to methods of using the preparations forthe treatment of a GD2 positive cancer in a patient.

The invention is further defined by the claims. All preferredembodiments of the invention as further described herein relate to allaspects of the invention equally and all of these aspects may becombined, e.g. the first with the second or third, the first with thesecond and third, the second with the third, to form a preparation for amethod of the combined elements of these aspects; or to form combinedmethods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of a WBT (with heparinized whole blood, using⁵¹Cr labeled target human neuroblastoma cells) and a CDC assay (withheparinized plasma, also using ⁵¹Cr labeled target human neuroblastomacells) of two healthy donors in the presence of APN311. As can be seen,there is a substantial difference in WBT lysis between the two donors:50% lysis is reached at APN311 concentrations of 2 versus 10 ng/mL wholeblood. However, there is no difference in CDC: 50% lysis of both donorsis reached at APN311 concentrations of 1000 ng/ml plasma. In both assays(WBT and CDC assay), the same incubation time (20 h) has been used, aswell as the same final concentration of complement.

FIG. 2 shows the results of a WBT (with heparinized whole blood, using⁵¹Cr labeled target human neuroblastoma cells) and a CDC assay (withheparinized plasma, using ⁵¹Cr labeled target human neuroblastoma cells)of one healthy donor in the presence of APN301 or APN311. There is asubstantial difference in WBT lysis between the two preparations: 50%lysis is reached at an APN311 concentration of 21 ng/mL whole bloodversus an APN301 concentration of 234 ng/mL. However, the difference inCDC is less substantial: 50% lysis is reached at an APN311 concentrationof 470 ng/mL plasma versus an APN301 concentration of 619 ng/mL plasma.

FIG. 3 shows the results of a WBT and CDC assay with whole blood orplasma of a healthy donor spiked with 5 μg/mL APN311 compared to thewhole blood or plasma of a patient treated with APN311. The patientsample was collected on day 17 of the treatment cycle, i.e. at the endof the treatment period with APN311, which in this case is from day 8 to18 of the treatment cycle.

FIG. 4 shows the results of the WBT as shown in FIG. 3 compared to thesame samples with the addition of a 5-fold excess of a specificanti-idiotypic (anti-ID) antibody, which inhibits the target cell lysis.

FIG. 5 shows the results of the CDC assay as shown in FIG. 3 compared tothe same samples with the addition of a 5-fold excess of specificanti-ID antibody, which inhibits the target cell lysis.

FIG. 6 shows the pharmacokinetics of APN311 in serum of patients. Thenumbers above the mean serum levels indicate the number of patientsincluded in said mean at this day of sample collection. The treatmentperiod with APN311 was from day 8 to 18, the two treatment periods withIL-2 were on days 1 to 5 and 8 to 12 of the treatment cycle.

FIG. 7 shows the CDC assay results on day 1, 8, and 15 of the treatmentcycle of 37 patients treated with APN311, as measured by a calceinrelease CDC assay. The treatment period with APN311 was from day 8 to18, the two treatment periods with IL-2 were on days 1 to 5 and 8 to 12.

FIGS. 8 and 9 show examples of schematic treatment schedules for thetreatment with a preparation comprising an anti-GD2 antibody combinedwith other treatments.

FIG. 10 shows the morphine use in % of the prescribed standard infusionrate (30 mcg/kg/h) during APN311 continuous infusions of 37 patients(mean values). Antibody infusions were always initiated on Day 8.

FIGS. 11 to 16 show cytolysis results obtained with blood samples frompatients who are in different stages of their treatment cycles. The dataare shown in a standardized format that represents the treatmentschedule as applied, namely APN311 in a dose of 100 mg/m²/cycle, 10 dayscontinuous infusion by mini-pump, i.v.; aldesleukin (IL2) in a dose of60×106 IU/m²/cycle, 10 days per cycle, administered in two 5-dayperiods, in a dose of 6×106 IU/m²/day s.c.; and 13-cis retinoid acid(isotretinoin) in a dose of 2240 mg/m²/cycle, administered for 14 days(once a day) in a dose of 160 mg/m²/day p.o. (per os, or oraladministration). The overall treatment time comprises 5 cyclescomprising 35 days per cycle, and day 36 is the first day of the secondtreatment cycle. The blood samples taken at the beginning (i.e. on thefirst day) of the treatment period with APN311 (corresponding to day 8of the treatment cycle) were taken prior to the start of the APN311treatment, see also table 8.

FIG. 17 shows the initial infusion rate of morphine administered duringthe antibody infusion in two different schedules (for the SIOPEN phase Itrial: 8 h antibody infusion for 5 subsequent days; for the continuousinfusion pilot schedule 24 h antibody infusion for 10 subsequent days),as well as the additional morphine administrations (given as a bolus)and the increases in the morphine infusion rate or the morphine dosethat were required.

FIG. 18 shows Kaplan-Meier curves of the event-free survival (EFS, FIG.18 A) and overall survival (OS, FIG. 18 B) data (in percent over time)of 328 neuroblastoma patients treated with APN311 and isotretinoin, butwithout IL-2 (in red) and patients treated with APN311 and isotretinoinand IL-2 (in blue).

FIG. 19 shows the comparison of the EFS data (in percent over time) of128 patients of Example 5 who had a complete response (CR) at start oftreatment (FIG. 19 A, the diagram on the right) with the EFS data (inpercent over time) of Yu et al. 2010, New England Journal of Medicine363:1324-1334 (FIG. 19 A, the diagram on the left, also comprising dataof complete responders). FIG. 19 B shows a chart overlay for comparison.

FIG. 20 shows the comparison of the OS data (in percent over time) of128 patients of Example 5 who had a complete response (CR) at start oftreatment (FIG. 20 A, the diagram on the right) with the OS data (inpercent over time) of Yu et al. 2010 (FIG. 20 A, the diagram on theleft). FIG. 20 B shows a chart overlay for comparison.

FIGS. 21 and 22 show overview tables of the toxicities (FIG. 21: allgrades of toxicities versus grade 3 and 4 only; FIG. 22: all grades oftoxicities for each treatment cycle) observed in percent of totalevaluated patients treated in the respective schedules with and withoutIL-2.

FIGS. 23 and 24 show the respective charts of toxicities (FIG. 23: allgrades of toxicities; FIG. 24: grades 3 and 4 only) observed in alltreatment cycles in percent of total evaluated patients treated in therespective schedules with and without IL-2.

FIGS. 25 and 26 show charts of all grade toxicities per treatment cyclein percent of total evaluated patients treated in the respectiveschedules with IL-2 (FIG. 26) and without IL-2 (FIG. 25).

FIGS. 27 to 32 show charts of toxicities observed in the respectivetreatment cycle (FIG. 27: cycle 1, FIG. 28: cycle 2, FIG. 29: cycle 3,FIG. 30: cycle 4, FIG. 31: cycle 5, FIG. 32: cycle 6) in percent oftotal evaluated patients treated in the respective schedules with andwithout IL-2.

FIG. 33 shows results of a complement-dependent cytotoxicity (CDC) assay(blue dots, FIG. 33 A) and a whole blood test (red dots, WBT, FIG. 33 B)of blood samples of a neuroblastoma patient during treatment with ananti-GD2 antibody, but without IL-2 and cis-retinioc acid treatment. Thepurple and grey dots are aliquots of patient samples treated with ananti-id antibody for differentiation of any potential non-specific lysis(i.e. target cell lysis that is not mediated by the antibody). Theorange bars indicate the treatment periods with the antibody.

FIG. 34 shows results of a complement-dependent cytotoxicity (CDC) assay(blue dots, FIG. 34 A) and a whole blood test (red dots, WBT, FIG. 34 B)of blood samples of a neuroblastoma patient during treatment with ananti-GD2 antibody and with a usual IL-2 dose and cis-retinioc acidtreatment. The purple and grey dots are aliquots of patient samplestreated with an anti-id antibody for differentiation of any potentialnon-specific lysis (i.e. target cell lysis that is not mediated by theantibody). The orange bars indicate the treatment periods with theantibody, the green triangles the IL-2 treatment periods, and the lightblue asterisks the cis-retinioc acid treatment periods.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, it has surprisingly turned out that a treatment witha preparation comprising an anti-GD2 antibody in a dose determined bycytolysis capacity, e.g. either measured by a CDC assay or by a WBT, hasa beneficial effect in cancer therapy, especially on side effects suchas pain. If the preparation comprising an anti-GD2 antibody isadministered in a dose as low as possible but sufficient to induce CDCand/or whole blood cytolysis, and is administered in said cytolysisthreshold dose for a treatment period until the predetermined overallpatient dose has been administered, pain can be substantially reducedand thus, the administration of morphine or other analgesics can besubstantially reduced or even stopped.

In this first aspect, the invention concerns a method for treating a GD2positive cancer by administering a preparation comprising an anti-GD2antibody to a patient as a continuous intravenous infusion over 24 hoursper day. The preparation comprising an anti-GD2 antibody may beadministered for a treatment period until the predetermined overallpatient dose has been administered.

Furthermore, the invention concerns a method for treating a GD2 positivecancer by administering a preparation comprising an anti-GD2 antibody toa patient, wherein the preparation is administered in a dose sufficientto induce tumor cell lysis (cytolysis threshold dose), and wherein saidcytolysis threshold dose is administered until the predetermined overallpatient dose has been administered.

In a second aspect, it has surprisingly turned out that treatment withone or more cytokines in combination with an anti-GD2 antibody and aretinoid does not provide any clinical benefit over the treatment withthe anti-GD2 antibody and a retinoid, but without any cytokine,especially without IL-2.

Thus, in this second aspect, the invention concerns a preparationcomprising an anti-GD2 antibody (also referred to as antibodypreparation) for use in the treatment of a GD2 positive cancer in apatient, wherein the preparation comprising an anti-GD2 antibody isadministered to the patient without concomitantly administering IL-2,and wherein one or more treatment periods with the antibody is/arepreceded, accompanied, and/or followed by one or more treatment periodswith a retinoid. Furthermore, the present invention concerns a methodfor treating a GD2 positive cancer by administering a preparationcomprising an anti-GD2 antibody to a patient, wherein the patient is notconcomitantly treated with Interleukin-2 (IL-2), and wherein one or moretreatment periods with the antibody is/are preceded, accompanied, and/orfollowed by one or more treatment periods with a retinoid.

The term “patient” as used herein shall mean an animal or human subjectsuffering from cancer, especially a GD2 positive cancer. In anembodiment, the patient is a human subject suffering from a GD2 positivecancer. The term “treatment” or “treating” as used herein shall meanthat a drug or treatment is administered to patient in need thereof.

The terms “concomitantly treated with” or “concomitantly administering”as used herein shall mean that one treatment (e.g. with an anti-GD2antibody and/or a preparation comprising an anti-GD2 antibody, referredto as antibody treatment) is preceded, accompanied, and/or followed bythe other one or more treatments (such as e.g. treatment with one ormore analgesics, and/or one or more other drugs or treatments), inparticular within the same treatment cycle and/or within the sameoverall treatment time (e.g. in which the anti-GD2 antibody isadministered). The treatment period of a concomitant treatment may ormay not overlap with the other treatment period (e.g. the antibodytreatment period), either partially or entirely. Accordingly, thetreatment period of a concomitant treatment (e.g. the analgesictreatment) may precede, accompany, and/or follow the treatment periodwith the other treatment (e.g. the antibody treatment period). In oneembodiment, the treatment periods of concomitant treatments are withinthe same treatment cycle.

Accordingly, the terms “not concomitantly treated with”, “withoutconcomitantly administering” or “not concomitantly administering” asused herein shall mean that one treatment is not preceded, accompanied,and/or followed by the one or more other treatments, respectively. Inone embodiment, the above defined terms shall mean that a patient is nottreated with said drug or treatment (i.e. that said drug or treatment isnot administered to said patient) within the same treatment cycle and/orwithin the same overall treatment time. Accordingly, the treatmentperiod of such a non-concomitant treatment (e.g. cytokine treatment) maynot overlap with the other treatment period (e.g. the antibody treatmentperiod), either partially or entirely. In an embodiment, the treatmentperiod of a non-concomitant treatment may not precede, accompany, and/oror follow the treatment period with the other treatment (e.g. theantibody treatment period). In one embodiment, the treatment periods ofnon-concomitant treatments are not within the same treatment cycle.However, a patient who is not concomitantly treated with a drug ortreatment (e.g. one or more cytokines) may have been treated with saiddrug or treatment (e.g. one or more cytokines) in previous treatmentcycles and/or previous overall treatment times.

The term “cytokines” as used herein shall mean proteins, peptides, orglycoproteins which act as hormonal regulators or signaling molecules atnanomolar to picomolar concentrations and help in cell signaling. In anembodiment, the one or more cytokines are selected from immunomodulatingagents, such as e.g. interleukins and/or interferons.

In an embodiment, the one or more cytokines are selected from the groupconsisting of IL-2, GM-CSF, Granulocyte colony-stimulating factor(G-CSF), IL-12, and/or IL-15. In one embodiment, the one or morecytokines are not fused to an antibody, in particular not to an anti-GD2antibody.

The term “reduced dose” or “low-dose” as used herein refers to a dose ofthe respective drug that is significantly lower, e.g. at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (or any range in betweenthese doses) lower than the usual dose of the same drug administered inthe same or similar setting, i.e. in the same or similar patient groupswith the same or similar treatment(s). The usual dose may be the dosethat has frequently been used in the past and/or is mainly used in thesame or similar setting, i.e. in the same or similar patient groups withthe same or similar treatment(s). The dose may be reduced by reducingthe daily dose, and/or by reducing the frequency and/or duration ofadministration. For example, for a 50% reduced dose, the respective drugmay be administered in the same frequency or duration as usual, but withonly half of the usual daily dose, or the respective drug may be givenin the usual daily dose, but e.g. only on every second day, if it hasusually been given every day. In another example, 50% of the usual dailydose may be given every second day instead of the usual dailyadministration, thus, resulting in a reduced dose that is 75% lower thanthe usual dose. Accordingly, a person skilled in the art can easilydetermine suitable doses and administration schedules according to theinvention.

A “treatment period” with a specific preparation or treatment as usedherein means the period of time in which said specific preparation ortreatment is administered to the patient within one treatment cycle,e.g. the time period of subsequent treatment days. For example, if thepreparation comprising a cytokine is usually administered for 5consecutive days, followed by one or more days of no administration ofthe preparation comprising a cytokine, then the treatment period withthe preparation comprising a cytokine comprises 5 days. In anotherexample, if the preparation comprising the anti-GD2 antibody isadministered continuously over 24 h for 10 consecutive days, followed byone or more days of no administration of the preparation comprising theanti-GD2 antibody, then the treatment period with the preparationcomprising the anti-GD2 antibody comprises 10 days. In another example,if isotretinoin is administered twice a day for 14 days, followed by oneor more days of no isotretinoin administration, then the treatmentperiod with isotretinoin comprises 14 days.

Any such treatment periods may be repeated, entirely or partiallyoverlap with other treatment periods with other drugs or treatments,and/or may be preceded and/or followed by periods of no treatment. Forexample, a treatment cycle (e.g. as depicted in FIGS. 8 and 9) maycomprise two 5-day treatment periods with IL-2, the second of which isoverlapping with a 10-day (or 14-, 15-, or 21-day) treatment period withch14.18 (APN311), followed by a 14-day treatment period withisotretinoin.

The terms “combined” or “combination” as used herein in relation totreatment periods shall mean that two or more treatment periods with thesame and/or different drugs or treatments are comprised in one treatmentcycle. Said two or more treatment periods with different drugs ortreatments may partially or entirely overlap, or may not overlap. Anysuch treatment periods may be combined with (or separated by) one ormore intervals of no treatment with the same and/or different drugs ortreatments.

The term “treatment cycle” as used herein means a course of one or moretreatments or treatment periods that is repeated on a regular schedule,optionally with periods of rest (no treatment) in between. For example,a treatment given for one week followed by three weeks of rest is onetreatment cycle. In one embodiment, one treatment cycle comprises onetreatment period with the preparation comprising an anti-GD2 antibody.The treatment cycle comprising one treatment period with the preparationcomprising an anti-GD2 antibody may further comprise one or moretreatment periods with one or more other drugs or treatments (for thesecond aspect of the invention except for cytokine treatment), such ase.g. retinoids, and/or analgesics. Any such treatment periods with oneor more drugs or treatments within one treatment cycle may entirelyand/or partially overlap. A treatment cycle may also comprise one ormore time periods without any treatment. The periods of rest may e.g. beat least 1 day, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, or 14 days ormore. Alternatively or in combination, the periods of rest may e.g. beat most 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1week or less. Each treatment in a treatment cycle may preferably be atreatment according to the first, second, third or any combined aspectas defined in the brief description.

The term “overall treatment time” as used herein shall mean thecontinuous treatment period comprising one or more subsequent treatmentcycles. A treatment cycle may be repeated, either identically or in anamended form, e.g. with a different dose or schedule, or with one ormore different and/or additional treatments (e.g. with one or more otheranalgesics). The overall treatment time may comprise at least 1, or 2 ormore cycles, e.g. up to 10 or up to 20 or even more treatment cycles. Inone embodiment, the overall treatment time comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 cycles. As described above, treatment cycles may comprisetime periods of no treatment (intervals in which no treatment isadministered to the patient, i.e. no antibody, no cytokine, and no otherdrug). Thus, as used herein, the overall treatment time may alsocomprise said intervals of no treatment within a treatment cycle and/orbetween treatment cycles. In one embodiment, a treatment cycle maydirectly follow after the previous treatment cycle, i.e. with no timeperiod in between treatment cycles. However, the end of a treatmentcycle may comprise a time period of no treatment, before the nexttreatment cycle begins. Example overall treatment times are e.g. atleast 6 months, 8 months, 10 months, 12 months, 14 months, 16 months, 18months, 20 months, 22 months, 24 months, 26 months, 30 months or more.

In some embodiments, the preparation comprising an anti-GD2 antibody isadministered to a patient in a dose sufficient to induce tumor celllysis (cytolysis threshold dose), and the preparation is administered asa continuous intravenous infusion over 24 hours per day. In otherembodiments, the preparation comprising an anti-GD2 antibody isadministered to a patient in a dose sufficient to induce tumor celllysis (cytolysis threshold dose), and the preparation is administered asa continuous intravenous infusion over 24 hours per day, and saidcytolysis threshold dose is administered until the predetermined overallpatient dose has been administered.

In certain embodiments, the cytolysis threshold dose is atherapeutically effective amount of the preparation comprising ananti-GD2 antibody. The therapeutically effective amount may bedetermined by a CDC assay or a WBT using patient's serum or plasma orheparinized whole blood. In some embodiments, the cytolysis thresholddose is a minimal cytolysis threshold dose, such as e.g. the lowest dosedetermined to induce a certain level of cytolysis in a CDC assay or aWBT. In one embodiment, the cytolysis threshold dose is the dosedetermined in a specific CDC assay or WBT to induce 30% of the maximalpossible target cell lysis in that respective assay. In certainembodiments, the cytolysis threshold dose is the dose that achieves 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or anyrange in between these levels, of the maximal possible cell lysis in therespective assay (a specific CDC assay or WBT). For example, as done inExamples 2 and 3 and as shown in FIGS. 1, 2 and 7, severalconcentrations of the preparation comprising the anti-GD2 antibody areeither spiked into the blood or plasma of the donor or already presentin the blood or plasma of the patient who has been treated with thepreparation comprising an anti-GD2 antibody, to determine a CDC or wholeblood lysis curve. By drawing a curve between the measuredconcentrations of anti-GD2 antibody, the dose or concentration ofanti-GD2 antibody achieving a certain threshold cytolysis (e.g. 50% ofthe maximal possible target cell lysis) can be determined. In theexample of FIG. 1, a threshold of 50% cytolysis (e.g. 50% of the maximalpossible target cell lysis) is achieved with concentrations of 2 or 10ng/mL whole blood of the respective donor in the WBT, or with 1000 ng/mLserum or plasma in the CDC assay. In this example, the thresholdcytolysis is 50%.

The terms “threshold cytolysis” and/or “level of cytolysis” as usedherein means the level of target cell lysis in a specific CDC assay orWBT specified to determine the cytolysis threshold dose in serum, plasmaor whole blood in said CDC assay or WBT.

In some embodiments, the threshold cytolysis is maintained even for oneor more time periods within the overall treatment time, where thepatient is not treated with the preparation comprising an anti-GD2antibody, i.e. in the intervals between the treatment periods with thepreparation comprising an anti-GD2 antibody (if any, i.e. if the patientis not treated continuously over the overall treatment time with thepreparation comprising an anti-GD2 antibody). In certain embodiments,the level of cytolysis is maintained over the entire treatment cycle. Insome embodiments, the level of cytolysis is maintained over the overalltreatment time.

As can be seen in FIGS. 11, 13, and 15, an increased level of cytolysisbetween 30% and 50% has been maintained even over the interval, wherethe patients have not been treated with the preparation comprising ananti-GD2 antibody.

In one embodiment, the cytolysis threshold dose is determinedindividually for each patient.

The term “predetermined overall patient dose” as used herein shall meanthe overall patient dose per treatment cycle, as further specifiedbelow.

If a range is given herein, any such range shall include any range inbetween the given ranges (i.e. the lower and the upper limit of therange). For example, if a range is given of e.g. 1 to 5 days, this shallinclude 1, 2, 3, 4, and 5 days. The same applies to any other ranges,including but not limited to other time periods (e.g. infusion time inhours), any dose ranges (e.g. per m² body surface area, per kg bodyweight, per day, per treatment cycle etc.), infusion rates,concentrations, percentages, factors, ratios, and numbers.

The cytolysis threshold dose may be determined by a complement dependentcytolysis (CDC) assay or a whole blood test (WBT). The WBT is an assayin which the target cells or target components (i.e. cells, liposomes orother cell-like compartments to be lysed) are contacted withappropriately anti-coagulated whole blood from the patient. The CDCassay can be, for example, a standard CDC assay as known in the art(e.g. as described in Indusogie et al., J Immunol 2000; Zeng et al.,Molecular Immunology 2005; or in WO2005/070967). The CDC assay and/orthe WBT may be done with GD2 positive target cells, such as tumor celllines of the GD2 positive cancer to be treated. For example, if thepatient to be treated suffers from neuroblastoma, the cell line may be aneuroblastoma cell line, such as e.g. LAN-1 human neuroblastoma cells.In another example, if the patient to be treated suffers from melanoma,the cell line may be a melanoma cell line, such as e.g. M21 humanmelanoma cells. In still another embodiment, the target cells of the CDCassay and/or the WBT are tumor cells obtained from the patient, i.e.autologous tumor cells of the patient. In another embodiment, the targetcomponent of the CDC assay and/or WBT is a liposome displaying GD2 onthe surface. The target cells or target components are labeled with asignaling component, e.g. with a radioactive component, such as ⁵¹Cr, orwith a fluorescent component, such as calcein. The signaling componentis comprised by the target cell or target component, i.e. is inside ofthe target cell or target component (e.g. a liposome packed with thesignaling component and displaying GD2 on the surface), and is releasedupon lysis of the target cell or target component. Thus, the signalingcomponent provides the assay readout. The target cells or componentsloaded with the signaling compound are contacted with the whole blood,serum, or plasma in a certain ratio. The whole blood, plasma, or serummay be diluted for the CDC or WBT, e.g. in a ratio of 1:2 or higher,e.g. 1:4, 1:5, or 1:10, or any range in between these ratios prior toadding it to the sample. However, it may also be added to the sampleun-diluted. The final concentration of the whole blood, plasma, or serumin the CDC or WBT sample may e.g. be in the range of 10 to 50%. Targetcell or target component lysis can be measured by release of saidsignaling component by a scintillation counter or spectrophotometry. Forexample, the target cell or target component lysis can be measured bydetermining the amount of ⁵¹Cr released into the supernatant by ascintillation counter. The percentage of lysis may be determined by thefollowing equation: 100×(experimental release−spontaneousrelease)/(maximum release−spontaneous release).

For the CDC assay, the cytolytic components (or effector components) areprovided by serum or appropriately anti-coagulated plasma obtained fromthe patient or donor comprising the complement system components. Forthe WBT, the cytolytic components (or effector components) are providedby appropriately anti-coagulated whole blood obtained from the patientor donor comprising the complement system components as well as allcellular components, and also any further components comprised in wholeblood which might be relevant to the target cell lysis, as well as theinterplay of all components (e.g. complement activation is known toactivate certain effector cells such as granulocytes). For the CDCand/or WBT, the serum, plasma, or whole blood may be added to the targetcells or target components in different dilutions.

Furthermore, one or more samples of the CDC assay and/or WBT may bespiked with an anti-GD2 antibody in different dilutions, e.g. forgeneration of a standard curve.

In another embodiment, one or more anti-idiotypic (anti-id) anti-GD2antibodies recognizing the variable domain of anti-GD2 antibodies may beadded to a sample to inhibit the target cell lysis mediated by theantibody, e.g. as a negative control or to prove specificity of theassay and that the target cell lysis measured without the anti-idantibody is antibody-mediated or antibody dependent.

If the cytolysis threshold dose is determined for a patient before thestart of the treatment with the preparation comprising an anti-GD2antibody, the anti-GD2 antibody or the preparation comprising theanti-GD2 antibody is added in different dilutions to the CDC assayand/or WBT samples (in addition to the patient serum, plasma, or blood),so that the cytolysis threshold dose can be determined.

As further described herein, target cells for determination of thethreshold dose may be human tumor cell lines of the same indication(e.g. human neuroblastoma cells in case of a neuroblastoma patient),or—if feasible—autologous tumor cells of the patient.

If the cytolysis threshold dose is determined for a patient during thetreatment with the preparation comprising an anti-GD2 antibody, theserum, plasma, or whole blood of the patient (which comprises theanti-GD2 antibody) is added in different dilutions to the CDC assayand/or WBT samples (without the addition of separate anti-GD2 antibody),so that the cytolysis threshold dose can be determined.

The dose sufficient to induce CDC and/or whole blood cytolysis may bedefined as the dose that achieves at least 20, 25, 30, 35, 40, 45, or50%, or any range in between these levels of the maximal possible targetcell lysis in that respective assay (a specific CDC assay or WBT). Inone embodiment, the dose is defined as the dose that achieves at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or 100%, or any range inbetween these levels of maximal possible cell lysis in the respectiveassay (a specific CDC assay or WBT).

The cytolysis threshold dose determined in a specific CDC assay or WBTis a serum-, plasma-, and/or blood-level of anti-GD2 antibody. The doseof the preparation comprising the anti-GD2 antibody to be administeredto patient to achieve such blood, plasma and/or serum antibody levelshas then to be determined accordingly based on pharmacokinetic data forsaid preparation. As shown in FIGS. 1 and 2, antibody levels as low as470 to 1000 ng/mL serum or plasma are sufficient to induce at least 50%tumor cell lysis in that CDC assay, e.g. 470 ng/mL (FIG. 2), or 1000ng/mL (FIG. 1) of APN311, and 619 ng/mL of APN301 (FIG. 2). Accordingly,in one embodiment of the invention, the cytolysis threshold dose is 470to 1000 ng/mL serum or plasma, or 470 to 10000 ng/mL serum or plasma, orany range in between these levels.

If a certain cytolysis threshold dose is determined in a CDC assay or aWBT, especially such assays in which target cells other than thepatient's tumor cells are used, said cytolytic threshold determined invitro (in vitro cytolytic threshold dose) may be increased by a certainmargin of safety to ensure that the antibody dose is sufficient toinduce cytolysis of the patient's tumor cells in vivo (in vivo cytolysisthreshold dose). Accordingly, the in vitro cytolysis threshold dose maybe increased by a factor of 1 to 10, or any range in between thesefactors.

In certain embodiments, the cytolysis threshold dose is 1410 to 3000ng/mL or 2350 to 5000 ng/mL serum or plasma, or any range in betweenthese levels.

The dose of the preparation comprising the anti-GD2 antibody to beadministered to the patient is determined accordingly, i.e. it isadministered in a dose to achieve said serum or plasma levels within thefirst 1-4 days of treatment with the preparation comprising the anti-GD2antibody (e.g. on day 1, 2, 3, or 4 of the treatment period with thepreparation comprising the anti-GD2 antibody), and said serum or plasmalevel is maintained over the entire treatment period with thepreparation comprising the anti-GD2 antibody. As shown in FIGS. 1 and 2,antibody levels as low as 2 to 234 ng/mL in whole blood are sufficientto induce at least 50% tumor cell lysis in that WBT, e.g. 2 ng/mL (FIG.1), or 10 ng/mL (FIG. 1), or 21 ng/mL (FIG. 2), of APN311, and 234 ng/mLof APN301 (FIG. 2). Accordingly, in one embodiment of the invention, thecytolysis threshold dose is 2 to 250 ng/mL whole blood, or 2 to 2500ng/mL whole blood, or any range in between these levels. In certainembodiments, the cytolysis threshold dose is 2 to 100 ng/mL whole blood,or 5 to 200 ng/mL whole blood, or any range in between these levels. Insome embodiments, the cytolysis threshold dose is 6 to 750, 6 to 7500,10 to 1250, 10 to 12500, 6 to 300, 10 to 500, 15 to 600, or 25 to 1000ng/mL whole blood.

The dose of the preparation comprising the anti-GD2 antibody to beadministered to the patient is determined accordingly, i.e. it isadministered in a dose to achieve said whole blood levels within thefirst 1-4 days of treatment with the preparation comprising the anti-GD2antibody (e.g. on day 1, 2, 3, or 4 of the treatment period with thepreparation comprising the anti-GD2 antibody), and said serum or plasmalevel is maintained over the entire treatment period with thepreparation comprising the anti-GD2 antibody. As can be seen in FIG. 6,serum levels of 1000 ng/mL (or 1 μg/mL) can be achieved within the firstone or two days of anti-GD2 antibody treatment, if the preparationcomprising the anti-GD2 antibody is administered in a dose of 10mg/m²/day as a continuous intravenous (i.v.) infusion, i.e. for 24 h perday, using a mini-pump. Thus, in one embodiment, the preparationcomprising the anti-GD2 antibody is administered in a dose of 5, 7, 10or 15, especially 10 mg/m²/day or any range in between these doses as acontinuous intravenous infusion (24 h per day). In one embodiment, thecytolysis threshold dose is achieved within the first, second, third orfourth day of the treatment with the preparation comprising the anti-GD2antibody. FIG. 7 shows that 50% of cytolysis can be achieved within thefirst three or four days of the treatment with the preparationcomprising the anti-GD2 antibody, if the preparation comprising theanti-GD2 antibody is administered in a dose of 10 mg/m²/day as acontinuous intravenous (i.v.) infusion, i.e. for 24 h per day, using amini-pump.

With the methods of the present invention it is possible to reduce theantibody dose to the minimum dose required for tumor cell lysis and/ortarget cell lysis as determined by a CDC assay or a WBT. In certainembodiments, the cytolysis threshold dose of the antibody determined bya CDC assay and/or a WBT is lower than 50, 40, 30, 25, 20, 15, 10, 7, 5mg/m²/day, or lower than any range in between these doses. Furthermore,the methods of the invention allow to individually determine thecytolysis threshold dose by a CDC assay and/or a WBT and thus, take intoaccount the individual differences in the lytic capacity against tumorcells of the patients. Accordingly, each patient may receive his or heroptimal antibody dose that is as low as possible to minimize potentialside effect, especially pain, but is effective in tumor cell lysis.

The antibody preparation may be administered to a subject or patient inneed thereof. In one embodiment, the subject or patient is a GD2positive cancer patient. A GD2 positive cancer is a type of cancer, inwhich GD2 is expressed on tumor cells and comprises, for example,neuroblastoma, glioblastoma, medulloblastoma, astrocytoma, melanoma,small-cell lung cancer, desmoplastic small round cell tumor,osteosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. In anembodiment, the patient has been diagnosed with neuroblastoma, inparticular high risk neuroblastoma. In one embodiment, the patient hasbeen diagnosed with stage 4 neuroblastoma (according to theInternational Neuroblastoma Staging System (INSS)). In an embodiment,the patient has been diagnosed with minimal residual disease. In anembodiment, the patient has been diagnosed as a complete responder, i.e.as a patient showing a complete response to treatment. In anotherembodiment, the patient has been diagnosed with relapsed or refractorydisease. In one embodiment, the patient suffers from primary refractoryor relapsed high risk-neuroblastoma, or from minimal residual disease inhigh-risk neuroblastoma. The patient may have previously been treated ormay be simultaneously treated with one or more other therapies, such ase.g. surgery, chemotherapy, radiation, myeloablative therapy,metaiodobenzylguanidine scintigraphy (mIBG), vaccine therapy, stem celltransplantation, cytokine treatment (e.g. with IL-2 and/or GM-CSF),and/or retinoid treatment (e.g. with isotretinoin).

In an embodiment, the patient is not enrolled in a clinical trial ofphase I, II or III. In another embodiment, the patient is not enrolledin any clinical trial. In particular, the patient is not enrolled in anyclinical trial in any country of the world. Accordingly, the patient isnot participating in any systematic investigation and/or officiallygranted (e.g. by any competent national or regional health authority)tests in medical research and drug development that generate safety andefficacy data for any drug or treatment. In an embodiment, the patientis not participating in any systematic investigation and/or officiallygranted (e.g. by any competent national or regional health authority)testing for any health interventions (including diagnostics, devices,etc.). The former clinical trials described in the prior art with ananti-GD2 antibody without concomitant treatment with IL-2 and/or anyother cytokine have been done to investigate general effects, adverseeffects, and doses of the antibody as a basis for further investigation.However, the prior art clearly teaches to finally treat patients with ananti-GD2 antibody in combination with at least one cytokine, especiallyIL-2. Accordingly, any currently used treatment regimes with an anti-GD2antibody comprise at least one cytokine, in particular IL-2, also incombination with GM-CSF (see e.g. Yu et al., cited above).

The antibody can be selected from the group of recombinant orartificial, including single chain antibodies, mammalian antibodies,human or humanized antibodies. It may comprise or be selected fromconstant and/or variable portions of an antibody in particular selectedfrom Fc, Fc-like, Fv, Fab, F(ab)₂, Fab′, F(ab′)₂, scFv, scfc, VHH.However, any such antibody fragment should comprise the Fc portion thatis responsible for complement binding, and thus, can mediate the natural(or in vivo) effector functions. Preferably the antibody comprises alight and heavy chain of an antibody. The antibody may comprise one ortwo antigen binding regions, which may bind the same or differentantigen, e.g. GD2, that may be bound specifically. The inventiveantibodies can be directed—e.g. generated by immunization against—theantigens as defined above. The anti-GD2 antibody may be a humanized orchimeric GD2 antibody, e.g. a humanized or chimeric 14.18, 3F8 or 8B6antibody, or a murine antibody with the same specificity, or anantigen-binding fragment of any of these which mediates the naturaleffector functions. In one embodiment, the antibody is not a 14G2aantibody. The anti-GD2 antibody may have one or more amino acidmodifications, such as e.g. a modified Fc region. In one embodiment, theanti-GD2 antibody is hu14.18K322A. In another embodiment, the anti-GD2antibody is a chimeric 14.18 antibody. In one embodiment, the anti-GD2antibody has the light chain nucleotide sequence of SEQ ID NO:1 (seealso Example 1) and the heavy chain nucleotide sequence of SEQ ID NO:2(see also Example 1). In one embodiment, the anti-GD2 antibody has thelight chain amino acid sequence of SEQ ID NO:3 (see also Example 1) andthe heavy chain amino acid sequence of SEQ ID NO:4 (see also Example 1).The relative molecular mass of the antibody comprising of two light andtwo heavy chains may be approximately 150,000 Dalton. In one embodiment,the preparation comprising the anti-GD2 antibody is APN311. The anti-GD2antibody may be expressed in CHO cells, in SP2/0 cells, or in othersuitable cell lines, such as e.g. HEK-293, MRC-5, Vero, PerC6, or NS0.In one embodiment, the anti-GD2 antibody is a chimeric 14.18 antibodyexpressed in SP2/0 cells (ch14.18/SP2/0). In another embodiment, theanti-GD2 antibody is a chimeric 14.18 antibody expressed in CHO cells(ch14.18/CHO).

In certain embodiments of the first aspect, the anti-GD2 antibody mayalso be an immunocytokine comprising a fusion protein of an anti-GD2antibody (or an antigen-binding fragment thereof which mediates thenatural effector functions) and a cytokine. The antibody part of theimmunocytokine may be a humanized or chimeric GD2 antibody, e.g. ahumanized or chimeric 14.18, 3F8 or 8B6 antibody. The antibody part ofthe immunocytokine protein may have one or more amino acidmodifications, such as e.g. a modified Fc region. In one embodiment, theantibody part of the immunocytokine is hu14.18K322A. In anotherembodiment, the antibody part of the immunocytokine is a humanized 14.18antibody. The cytokine part of the anti-GD2 antibody-cytokine fusionprotein may be, for example, IL-2 or Interleukin-12 (IL-12), or IL-15 orGM-CSF. The antibody and the cytokine are fused together and maycomprise a linker sequence. In one embodiment, the immunocytokine hasthe light chain nucleotide sequence of SEQ ID NO:5 (see also Example 1)and the heavy chain nucleotide sequence of SEQ ID NO:6 (see also Example1). In one embodiment, the immunocytokine has the light chain amino acidsequence of SEQ ID NO:7 (see also Example 1) and the heavy chain aminoacid sequence of SEQ ID NO:8 (see also Example 1). In one embodiment,the immunocytokine is APN301. The immunocytokine may be expressed in NS0cells, or in other suitable cell lines, such as e.g. CHO, HEK-293,MRC-5, Vero, or PerC6.

In certain embodiments, the anti-GD2 antibody is not fused to any othermoiety. In certain embodiments, the anti-GD2 antibody is not animmunocytokine. In certain embodiments, the preparation comprising ananti-GD2 antibody does not comprise an immunocytokine. In certainembodiments, the patient is not concomitantly treated with animmunocytokine, in particular not within the same treatment cycle and/orwithin the same overall treatment time comprising the antibodytreatment.

The preparation comprising an anti-GD2 antibody may further comprisesalts and WFI, and optionally amino acids, in particular basic aminoacids, such as e.g. histidine, arginine and/or lysine. In oneembodiment, the preparation comprising an anti-GD2 antibody may furthercomprise a buffer, e.g. phosphate buffered saline, comprising said saltsand WFI.

The preparation comprising an anti-GD2 antibody may further comprisestabilizing agents, preservatives and other carriers or excipients. Thepreparation comprising an anti-GD2 antibody may be freeze-dried. In oneembodiment according to the first aspect of the invention, thepreparation comprising an anti-GD2 antibody comprises an anti-GD2antibody-cytokine fusion (e.g. hu14.18-IL-2) and further comprisessucrose, L-arginine, citric acid monohydrate, polysorbate 20, andhydrochloric acid. In an embodiment, the preparation comprising ananti-GD2 antibody is APN301, the anti-GD2 antibody is hu14.18-IL-2 andthe preparation comprises 4 mg/mL immunocytokine, 20 mg/mL sucrose, 13.9mg/mL L-arginine, 2 mg/mL polysorbate 20, and 2.1 mg/mL citric acidmonohydrate. In an embodiment, said preparation comprising animmunocytokine and other excipients is freeze-dried, can bereconstituted in 4 mL of 0.9% sodium chloride, and the resultingsolution has a pH of 5.5 (pH can be adjusted with hydrochloric acid(HCL)). In an embodiment, the preparation comprising an anti-GD2antibody is APN311, the anti-GD2 antibody is ch14.18/CHO and thepreparation further comprises sucrose, polysorbate 20, histidine, andhydrochloric acid. In an embodiment, the antibody is ch14.18/CHO, thepreparation comprising the antibody is APN311 (in an amendedformulation), and said preparation comprises 4.5 mg/mL antibody, 50mg/mL sucrose, 0.1 mg/mL polysorbate 20, and 3.1 mg/mL histidine. Thepreparation comprising an anti-GD2 antibody may be freeze-dried. Thereconstituted solution may have a pH of 6±0.5. In one embodiment, thepreparation comprising an anti-GD2 antibody does not comprisestabilising agents, preservatives and other excipients. The preparationcomprising an anti-GD2 antibody may be added to an infusion bag, e.g. aninfusion bag containing normal saline, i.e. a physiologic NaCl solution(0.9%), optionally with human serum albumin (HAS, also referred to ashuman albumin). In an example, the infusion bag comprises normal salineand 0.25-5% human serum albumin, or any range in between theseconcentrations. In an example, the infusion bag comprises a final volumeof 250 ml NaCl 0.9% and 5 ml human albumin 20%, or 100 ml NaCl 0.9% and5 ml human albumin 20%, or 50 ml NaCl 0.9% and 2 ml human albumin 20%.

The anti-GD2 antibody or the preparation comprising an anti-GD2 antibodymay be administered in daily antibody doses of 1 to 30 mg/m², 1 to 35mg/m², 1 to 50 mg/m², or 1 to 60 mg/m², e.g. 1, 2, 3, 4, 5, 6, 7, 7.5,8, 9, 10, 12, 15, 20, 25, 30, 32, 35, 40, 45, 50, or 60 mg/m² or anyrange in between these periods. For example, a daily dose of 10 mg/m²means that the patient receives 10 mg anti-GD2 antibody per m² of bodysurface per day. As used herein, a dose (e.g. given in mg or microgram)refers to the dose of the active ingredient, i.e. to the amount ofactive ingredient in the preparation. For example, the given dose mayrefer to the amount of anti-GD2 antibody in the preparation comprisingan anti-GD2 antibody, or the immunocytokine in the preparationcomprising the immunocytokine, or the cytokine in the preparationcomprising the cytokine, or the morphine or other analgesic in thepreparation comprising morphine or other analgesics etc. As specified inthe example above, a daily dose of 10 mg/m² means that the patientreceives 10 mg anti-GD2 antibody (optionally contained in a certainvolume of the preparation comprising the anti-GD2 antibody) per m² ofbody surface per day. As used herein, a dose given per m² means per m²of body surface area (BSA) of the patient. As used herein, a dose givenper kg means per kg of body weight of the patient.

In some embodiments, the preparation comprising an anti-GD2 antibody isadministered in daily doses of 1 to 15, 1 to 20, 1 to 25, 1 to 30, or 1to 35 mg/m², or any range in between these daily doses. In certainembodiments, the preparation comprising an anti-GD2 antibody isadministered in daily doses of less than 50, 40, 30 or 25 mg/m². Incertain embodiments, the preparation comprising an anti-GD2 antibody isadministered in daily doses of up to 7, 10, 15, or 20 mg/m². In oneembodiment, the antibody preparation is administered in a dose of 25mg/m²/day for 4 days. In one embodiment, the antibody preparation isadministered in a dose of 50 mg/m²/day for 4 days. The anti-GD2 antibodymay be administered in a dose of 10, 20, 25, 50, 60, 75, 80, 100, 120,150, 200, 210, 250, or 300 mg/m²/cycle or any range in between thesedoses. The total dose per patient per treatment cycle may be defined asthe predetermined overall patient dose.

In some embodiments, especially in embodiments of the second aspect ofthe invention, the preparation comprising an anti-GD2 antibody isadministered as an intravenous infusion over 5 h or more per day, e.g.5.75 h or more per day, 8 h or more per day, 10 h or more per day, or upto 20 h per day, or any range in between these time periods, e.g. for 4or 5 days or more. In other embodiments, the preparation comprising ananti-GD2 antibody is administered as a continuous intravenous infusionover 24 h per day, e.g. for 10, 14, 15, or for up to 21 or more days.

In some embodiments, the preparation comprising an anti-GD2 antibody isadministered for a treatment period until a certain therapeutic effecthas been reached. In some embodiments, the therapeutic effect may be anincrease in immune response to the tumor, as determined, for example, byan increase in immune system biomarkers (e.g. blood parameters, such aslymphocyte counts and/or NK cell numbers; and/or cytokines). In someembodiments, the therapeutic effect may be a reduction in tumor markers(e.g. catecholamines). In some embodiments, the therapeutic effect maybe determined by methods such as metaiodobenzylguanidine scintigraphy(mIBG), magnetic resonance imaging (MRI) or X-ray computed tomography(CT), and/or bone marrow histology (assessed by aspirate or trephinebiopsy), and/or CDC assays and/or WBTs.

In certain embodiments, the therapeutic effect may be defined as stabledisease (i.e. no further increase in lesions, tumor tissue and/or size),partial response (i.e. reduction in lesions, tumor tissue and/or size),and/or complete response (i.e. complete remission of all lesions andtumor tissue.

Complete Response (CR) may be further defined as follows:

-   -   Complete disappearance of all measurable and evaluable disease;    -   no new lesions;    -   no disease-related symptoms; and/or    -   no evidence of evaluable disease, including e.g. normalization        of markers and/or other abnormal lab values.

In some embodiments, all measurable, evaluable, and non-evaluablelesions and sites must be assessed using the same technique as baseline.

Partial Response (PR) may be further defined as follows:

-   -   Applies only to patients with at least one measurable lesion;    -   Greater than or equal to 50% decrease under baseline in the sum        of products of perpendicular diameters of all measurable        lesions;    -   No progression of evaluable disease;    -   No new lesions.

In some embodiments, all measurable and evaluable lesions and sites mustbe assessed using the same techniques as baseline.

The preparation comprising an anti-GD2 antibody may be administered asdescribed in WO2013/189516 or WO2013/189554. For example, thepreparation comprising an anti-GD2 antibody may be administered ascontinuous intravenous infusion for 24 hours per day. Accordingly, in anembodiment the invention relates to a preparation comprising an anti-GD2antibody for use in the treatment of a GD2 positive cancer in a patient,wherein the preparation comprising an anti-GD2 antibody is administeredto the patient as a continuous infusion for one or more days and for twoor more treatment cycles, without concomitantly administering IL-2. Inanother embodiment, the invention relates to a preparation comprising ananti-GD2 antibody for use in the treatment of a GD2 positive cancer in apatient, wherein the preparation comprising an anti-GD2 antibody isadministered to the patient as a continuous infusion withoutconcomitantly administering IL-2, and wherein the anti-GD2 antibody isnot a 14G2a antibody.

The preparation comprising an anti-GD2 antibody may be administered for4, 5, 10, 14, 15, or 21 consecutive days or any range in between theseperiods. The preparation comprising an anti-GD2 antibody may also beadministered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, or more consecutive days. In certain embodiments,the preparation comprising an anti-GD2 antibody is administered over theentire treatment cycle, e.g. for 35 days. In some embodiments, thepreparation comprising an anti-GD2 antibody is administered as acontinuous intravenous infusion for the overall treatment time, e.g.over 5 treatment cycles with 35 days each, i.e. over 180 days in total.The daily antibody dose may be reduced accordingly, so that thepredetermined patient dose of the antibody is administered. In oneembodiment, the predetermined patient dose of the antibody is 100mg/m²/cycle. In one embodiment, the overall treatment time comprises 5cycles. Accordingly, in this example, the antibody dose per overalltreatment time is 500 mg/m². In an embodiment, this total antibody doseof 500 mg/m² per overall treatment time is administered over 180 days,i.e. in 2.77 mg/m²/day. The preparation comprising an anti-GD2 antibodymay be administered as a continuous intravenous infusion over a timeperiod of 24 hours per day. For such continuous infusion, an osmoticmini-pump may be used. In one embodiment, the preparation comprising ananti-GD2 antibody is administered as continuous intravenous infusion for24 hours per day for 4, 5, 10, 14, 15 or 21 consecutive days or anyrange in between these periods, in daily doses as specified above (e.g.7, 10, 15, 20, or 25 mg/m²/day), e.g. 25 mg/m²/day for 4 days, 20mg/m²/day for 5 days, 10 mg/m²/day for 10 days, 15 mg/m²/day for 10days, 7 mg/m²/day for 14 days, 15 mg/m²/day for 14 days, 10 mg/m²/dayfor 15 days, 7 mg/m²/day for 21 days, or 10 mg/m²/day for 21 days or anyrange in between these doses. In certain embodiments, the preparationcomprising an anti-GD2 antibody is not administered as continuousintravenous infusion for 5 days in a daily dose of 40 mg/m². In certainembodiments, the preparation comprising an anti-GD2 antibody is notadministered as continuous intravenous infusion for 5 days, i.e. not asa 120-hour-infusion. In some embodiments, the preparation comprising ananti-GD2 antibody is administered as continuous intravenous infusion formore than 5 days. In some embodiments, the preparation comprising ananti-GD2 antibody is administered as continuous intravenous infusion for6 or more days.

In one embodiment, the preparation comprising the anti-GD2 antibody isAPN311 and is administered in a dose of 10 mg/m²/day for 10 days. In oneembodiment, the preparation comprising the anti-GD2 antibody is APN311and is administered in a dose of 10 mg/m²/day for 10 consecutive daysfor 1, 2, 3, 4, 5, or 6 or more treatment cycles.

According to the second aspect of the present invention, the patient isnot concomitantly treated with IL-2, GM-CSF, and/or another cytokine. Inan embodiment, the antibody treatment period is not preceded,accompanied, and/or followed by one or more treatment periods with IL-2,GM-CSF, and/or one or more other cytokines, i.e. the preparationcomprising an anti-GD2 antibody is administered to the patient withoutconcomitantly administering IL-2, GM-CSF, and/or one or more othercytokines. In one embodiment, the treatment period with the preparationcomprising an anti-GD2 antibody is not accompanied by one or moretreatment periods with a cytokine (or a preparation comprising one ormore cytokines), in particular not within the same treatment cycleand/or within the same overall treatment time (comprising the antibodytreatment). For example, the patient is not concomitantly treated withGranulocyte colony-stimulating factor (G-CSF), GM-CSF, IL-2, IL-12,and/or IL-15. In an embodiment, the preparation comprising an anti-GD2antibody is administered to the patient without concomitantlyadministering IL-2. In an embodiment, the preparation comprising ananti-GD2 antibody is administered to the patient without concomitantlyadministering GM-CSF. In an embodiment, IL-2 and/or GM-CSF (or apreparation comprising IL-2 and/or GM-CSF) is not administered to thepatient within the same treatment cycle that comprises the antibodytreatment period(s). In an embodiment, the preparation comprising ananti-GD2 antibody is administered to the patient without concomitantlyadministering IL-2 and/or GM-CSF. In an embodiment, the preparationcomprising an anti-GD2 antibody is administered to the patient withoutconcomitantly administering a cytokine.

The treatment period with the preparation comprising an anti-GD2antibody may be preceded, accompanied, and/or followed by one or moretreatment periods with a retinoid (or a preparation comprising aretinoid). In an embodiment, the one or more treatment periods with thepreparation comprising an anti-GD2 antibody is/are followed by one ormore treatment periods with a retinoid. Said one or more treatmentperiods with a retinoid may follow within the same one or more treatmentcycles comprising one or more antibody treatment periods, or thereafter,i.e. after the end of the one or more treatment cycles comprising theone or more antibody treatment period, in particular, after the end ofthe last treatment cycle comprising one or more antibody treatmentperiods, i.e. at the end of the overall treatment time with theantibody. Accordingly, in an embodiment, there is a time period inbetween the antibody treatment period and the retinoid treatment period,which may be a time period of no treatment, or may comprise one or moretreatment periods with one or more other drugs or treatments. In oneembodiment, the retinoid is a retinoic acid (RA), e.g. isotretinoin(cis-RA). The retinoid may be a first generation retinoid (e.g. retinol,retinal, tretinoin (retinoic acid, Retin-A), isotretinoin, andalitretinoin), a second generation retinoid (e.g. etretinate and itsmetabolite acitretin), and/or a third generation retinoid (e.g.tazarotene, bexarotene and Adapalene). The retinoid may also be aretinoid derivative, in particular a synthetic retinoid derivative, suchas e.g. fenretinide. The preceding or following one or more treatmentperiods with a retinoid may be a treatment period without antibodyand/or IL-2 (or other cytokine) administration.

In certain embodiments of the first aspect of the invention, theimmunocytokine or the preparation comprising the immunocytokine may beadministered in daily immunocytokine doses of 0.8 to 50 mg/m², e.g. 0.8,1.6, 2, 3.2, 4, 4.8, 5, 6, 7, 7.5, 8, 9, 10, 12, 14.4, 15, 20, 25, 30,32, 40, 45, or 50 mg/m² or any range in between these doses. Forexample, a daily dose of 10 mg/m² means that the patient receives 10 mgimmunocytokine per m² of body surface per day. In one embodiment, onemilligram of fusion protein contains approximately 0.8 mg of hu14.18antibody and approximately 3×10⁶ U of IL-2. The preparation comprisingan immunocytokine may be administered subcutaneously or as intravenousinfusion, e.g. once a day. The preparation comprising an immunocytokinemay be administered i.v. over a time period of 24 hours per day. Thepreparation comprising an immunocytokine may be administered for 2, 3,4, 5, 10, 14, 15, or 21 consecutive days or any range in between theseperiods. In another embodiment, the preparation comprising animmunocytokine is administered as continuous intravenous infusion for 24hours per day for 10, 14, 15 or 21 consecutive days. For such continuousinfusion, an osmotic mini-pump may be used. In one embodiment, theimmunocytokine is administered in a dose of 12 mg/m²/day for 3consecutive days in 28-day cycles (in up to 10 cycles).

In the first aspect of the invention, the treatment period with thepreparation comprising an anti-GD2 antibody may be preceded and/oraccompanied by one or more treatment periods with a cytokine. In oneembodiment, the cytokine is Granulocyte colony-stimulating factor(G-CSF), GM-CSF, IL-2, IL-12, and/or IL-15. The cytokine may beadministered subcutaneously (e.g. once a day), or as intravenousinfusion. In one embodiment, the cytokine is IL-2 and is administeredsubcutaneously once a day in a dose of 6×10⁶ IU/m²/day, e.g. on days 1and 2 and on days 8 to 14 of the treatment cycle, or e.g. on days 1 to 5and on days 8 to 12 of the treatment cycle. In one embodiment, theoverall patient dose of IL-2 is 60×10⁶ IU/m²/cycle. In anotherembodiment, the cytokine is GM-CSF and is administered intravenouslyonce a day over 2 hours in a dose of 250 micrograms/m²/day, e.g. on days1 and 2 and 8 to 14 of the treatment cycle.

As described herein, the treatment period with the preparationcomprising an anti-GD2 antibody may be followed by one or more treatmentperiods with a retinoid. In one embodiment, the retinoid is a retinoicacid (RA), e.g. isotretinoin.

Any such treatment period may be repeated. Any such treatment period maybe followed by an interval of no treatment, either with the same and/orwith different drugs or treatments. In one embodiment, the interval maybe an interval free of any treatment. In another embodiment, theinterval is free of administration of the same preparation or treatment,however, other preparations or treatments may be administered during theinterval.

Furthermore, the treatment according to the present invention may bepreceded and/or accompanied by a treatment with one or more analgesics(or a preparation comprising one or more analgesics), such as e.g.non-steroidal anti-inflammatory drugs (NSAIDs, e.g. indometacin), and/orone or more opioids, and/or one or more other analgesics, or anycombination thereof. In one embodiment, the analgesic is an opioid, e.g.morphine and/or morphine derivatives, such as e.g. hydromorphone. Otheropioids are, for example, tramadol, pethidine, codeine, piritramide,levomethadone, as well as fentanyl, alfentanil, remifentanil andsufentanil.

As described above, an anti-GD2 antibody has usually been administeredto a patient in combination with one or more cytokines (in the usualdoses), especially IL-2 and/or GM-CSF. However, the combination of theantibody treatment with the usual cytokine treatment has potentiatedside effects of the antibody treatment, in particular pain. Thus, thetreatment with one or more analgesics, especially morphine, wasrequired. It has now surprisingly turned out that anti-GD2 antibodytreatment is efficient even without cytokine treatment. Accordingly, bythe total omission of any cytokine treatment the pain side effect (andother side effects) can be significantly reduced and thus, theadministration of morphine and/or other analgesics can be reduced aswell.

The dose of analgesics can be further reduced by a long-term infusion ofthe anti-GD2 antibody, e.g. as continuous i.v. infusion over 24 h perday, as described in the first aspect of the invention and inWO2013/189516 or WO2013/189554. Thus, the analgesic dose can besubstantially reduced in an inventive treatment schedule with nocytokine(s) together with a continuous infusion of the antibody.

With the preparations and methods of the invention, it is possible toreduce the dose, to change the route of administration (e.g. fromintravenous infusion to oral), to reduce the duration of the analgesictreatment period(s), and/or to change the kind of preparation of the oneor more analgesics. Thus, the present invention even allows for anoutpatient management, at least for a part of the treatment cycle, ofpatients on treatment with a preparation comprising an anti-GD2antibody.

The one or more analgesics may be administered orally. The one or moreanalgesics may also be administered as intravenous infusion, especiallyas continuous intravenous infusion for 24 hours per day. The treatmentperiod with the one or more analgesics may precede and/or accompany thetreatment period with the preparation comprising an anti-GD2 antibody.

In some embodiments, the one or more analgesics may be selected fromGABA-analogues, such as e.g. gabapentin. Accordingly, the patient may betreated with gabapentin, e.g. three days prior to the start of theantibody treatment period.

The following doses and examples of analgesic treatment are usual dosesthat may be reduced as described herein. Usually, in this settingGabapentin is administered orally in a dose of 10 mg/kg/dose once, twiceor three times a day. Gabapentin may be given in a dose of up to 300mg/kg/dose. Gabapentin is available and may be administered as oralsolution containing 250 mg/5 mL of gabapentin, or in capsules (100 mg,300 mg, and 400 mg). The gabapentin treatment may be administeredinstead of or in addition to the treatment with morphine and/or otheranalgesics. Furthermore, the patient may be treated with paracetamol (10to 15 mg/kg/dose, every 4 hours or four times a day, orally orintravenously), ibuprofen (5 to 10 mg/kg/dose orally every 6 to 12hours), metamizol (10 to 15 mg/kg/dose orally every 4 hours),diphenhydramine (0.5 to 1 mg/kg/dose orally or intravenously), and/orindometacin (e.g. 0.3 to 0.5 mg/kg/dose, or 25 or 50 mg/dose, orally orintravenously every 6 hours). Said treatment with paracetamol,ibuprofen, metamizol, and/or indometacin may be administered instead ofor in addition to the treatment with morphine and/or gabapentin, and/orother analgesics.

In some embodiments, the daily dose of the one or more analgesics on oneor more antibody treatment days according to the invention (continuousinfusion) is lower than the usual daily dose administered during thetreatment with a preparation comprising an anti-GD2 antibody that isadministered as a non-continuous intravenous infusion, or that isadministered as a continuous intravenous infusion for 5 days in a dailydose of 40 mg/m².

In certain embodiments, the dose (e.g. the daily dose) of the one ormore analgesics (e.g. morphine) is reduced over time, e.g. within theoverall treatment time, within a treatment cycle, during the antibodytreatment period within a treatment cycle, from one antibody treatmentday to the next antibody treatment day within a treatment cycle, and/orfrom one treatment cycle to the next. Examples of such morphine dosereductions are given in Table 9. For example, the morphine dose can bereduced by 10% from day 9 to 10 of the third treatment cycle, namelyfrom 28% to 18% of the standard infusion rate (which in this example is30 mcg/kg/h), or from 8.1 to 4.53 mg/kg/h, or from 0.19 to 0.11mg/kg/day. In some embodiments, the morphine dose is continuouslyreduced within a treatment cycle, during the antibody treatment periodwithin a treatment cycle, and/or from one antibody treatment day to thenext antibody treatment day within a treatment cycle.

In some embodiments, morphine is used as analgesic, optionally incombination with one or more other analgesics.

For example, such usual morphine doses administered before and/or duringa non-continuous infusion (or bolus infusion, i.e. an infusion for lessthan 24 hours a day) treatment period with a preparation comprising ananti-GD2 antibody are given in table 1. In this example of usual doses,the ch14.18/CHO (APN311) is given as an 8 hour infusion per day on 5subsequent days in a dose of 20 mg/m²/day and thus 100 mg/m²/cycle, andIL-2 is given s.c. in 6 MIU/m²/day for 5 consecutive days two times percycle (i.e. 60 MIU/m²/cycle), for 5 cycles, and morphine hydrochlorideis given on each antibody treatment day (i.e. each day of a treatmentcycle on which the preparation comprising an anti-GD2 antibody isadministered to the patient) in a bolus dose of 0.05 mg/kg/h for 2 hoursprior to starting the APN311 infusion, in an infusion rate of 0.03mg/kg/h for 8 hours during the APN311 infusion, and in an intervalinfusion rate of 0.01 mg/kg/h for 14 hours on the first day of APN311treatment, and for 4 hours on subsequent treatment days, if tolerated(with an interval of 10 hours with no morphine treatment). The dose wasincreased (e.g. increase in infusion rate during antibody infusion)and/or additional bolus doses were administered on an as needed basis.Accordingly, the prescribed morphine dose was at least 0.38 mg/kg perday, at least 2 mg/kg per treatment cycle (comprising 5 antibodytreatment days), and at least 10 mg/kg per overall treatment time(comprising three cycles).

In certain embodiments, the one or more daily morphine doses and/or theone or more morphine infusion rates and/or the one or more percentagesof the standard morphine doses are as specified in Table 9. For example,in one embodiment the percentage of the standard morphine doseadministered on day 12 of the first treatment cycle is 41%, the morphineinfusion rate on day 12 of the first treatment cycle is 12.26 mg/kg/h,and the daily morphine dose on day 12 of the first treatment cycle is0.29 mg/kg.

TABLE 1 Morphine infusion schedule Prepare 10 mg morphine in 40 mLglucose 5% (0.25 mg = 1 mL) duration of morphine morphine morphineinfusion infusion rate dose (h) (mg/kg/h) mg/kg pre-infusion 2 0.05 0.1 infusion during APN311 8 0.03 0.24 treatment interval infusion 14 or 40.01 0.14 or 0.04 total dose per treatment 0.48 or 0.38 day (mg/kg/24 h)

In another example of usual doses, APN311 is given as an 8 hour infusionper day on 5 subsequent days for 3 cycles in a dose of 10, 20, and 30mg/m²/day and 50, 100, 150 mg/m²/cycle, and IL-2 is given s.c. in 6MIU/m²/day for 5 consecutive days two times per cycle (i.e. 60MIU/m²/cycle), and morphine hydrochloride is given on each antibodytreatment day in a bolus dose of 0.5-1.0 mg/kg/dose (just prior to thestart of infusion of the antibody), and in a rate of 0.05 mg/kg/hourcontinuous infusion during the APN311 infusion. The dose is increased(e.g. increase in infusion rate during antibody infusion) and/oradditional bolus doses are administered on an as needed basis.Accordingly, the prescribed morphine dose (or usual morphine dose) is atleast 0.9 mg/kg per day, at least 4.5 mg/kg per treatment cycle(comprising 5 antibody treatment days), and at least 13.5 mg/kg peroverall treatment time (comprising three cycles).

In other examples of usual treatment with non-continuous (or bolus)infusions of ch14.18 and one or more cytokines, morphine has beenadministered in infusion rates up to 1.2 mg/kg/h over 24 hours.

In still another example of usual doses, ch14.18/Sp2/0 is given in adose of 25 mg/m²/day on four consecutive days. In one embodiment, Eachdose of ch14.18/Sp2/0 is infused i.v. over 5.75 or more (maybe extendedto up to 20 h), or 10 hours or more (maybe extended to up to 20 h),starting at 1.25 mg/m²/h×0.5 h, then 2.5 mg/m²/h×0.5 h, and optionallyfurther increasing the infusion rate to then 3.75 mg/m²/h×0.5 h, andthen to 5 mg/m²/h for the remaining dose, if tolerated. IL-2 is givenevery second cycle (e.g. cycle 2 and 4) as a continuous i.v. infusionover 4 days (96 h) in 3 MIU/m²/day in week 1 of the treatment cycle andin 4.5 MIU/m²/day in week 2 of the treatment cycle (i.e. 30MIU/m²/cycle). GM-CSF is given s.c. in a daily dose of 250 mcg/m². In anembodiment, each treatment cycle starts on day 0, and day 0 of atreatment cycle is the first day of treatment with the respectivecytokine. The overall treatment time may comprise 1, 2, 3, 4, or 5 ormore treatment cycles. Tables 2, 3, and 4 show examples of usualtreatment schedules.

TABLE 2 Schema for the usual administration of 5 cycles ofch14.18/Sp2/0, cytokines, and isotretinoin (retinoic acid or RA). Cycle1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Ch14.18 Ch14.18 Ch14.18 Ch14.18Ch14.18 GM-CSF Aldesleukin GM-CSF Aldesleukin GM-CSF (IL-2) (IL-2) RA RARA RA RA

In said example of usual doses, Ch14.18/SP2/0 treatment is administeredevery 28 days at 25 mg/m²/day×4 days for all 5 cycles; GM-CSF at 250micrograms/m²/day for 14 days; Aldesleukin (IL-2) at 3 MIU/m²/day forfirst week, and at 4.5 MIU/m²/day for second week.

TABLE 3 Treatment schema for cycles with GM-CSF Day 0 1 2 3 4 5 6 7 8 910 11 12 13 14-23 24 GM-CSF X X X X X X X X X X X X X X Begin ch14.18

Cycle 2&4 RA

Note: In variation to the treatment schema above, the RA treatment isstarted on day 11 of the first cycle, but according to the schema on day10 of the third and fifth cycle. Accordingly, in variation to thetreatment schema above, day 24 of the first treatment cycle is also thelast day of RA treatment of the first cycle.

In said example of usual doses, GM-CSF is given at 250 micrograms/m²/dayas subcutaneous injection (strongly recommended) or i.v. as a 2 hourinfusion daily from Day 0 through 13 (daily with the infusion ofch14.18/SP2/0 and for 3 days before and 7 days after the antibodytreatment).

TABLE 4 Treatment schema for cycles with aldesleukin (IL-2) Day 0 1 2 34-6 7 8 9 10 11-13 14 15 16 17 18-27 IL-2 X X X X X X X X ch14.18

RA

On days 28-31 of the aldesleukin cycles no treatment is administered. Onday 32, the next treatment cycle (with GM-CSF) is started (day 32=day 0of the following cycle).

As an example of usual doses, Aldesleukin (Interleukin-2, IL-2) may begiven in a dose of 3 MIU/m²/day by continuous infusion (using a CADD®Ambulatory Infusion Pump or a similar infusion pump) for 4 days (on Days0-3) during the first week of each cycle. During the second week of eachcycle, Aldesleukin (IL-2) may be given at 4.5 MIU/m²/day for 4 days (onDays 7 to 10, with the infusion of ch14.18/SP2/0). Aldesleukin may becontinuously infused i.v. over 96 hours through a catheter via anambulatory infusion pump in 5% dextrose in water (may contain 0.1% humanserum albumin if needed), total volume dependent upon the pump.

A sixth treatment cycle may be added with 14 days of no treatment(starting on day 24 of the fifth cycle, which is day 0 of the sixthcycle) followed by 14 days of the administration of isotretinoin only.

In an example of usual doses in this setting (i.e. with the treatmentsas described above), hydroxyzine (1 mg/kg; max dose 50 mg) ordiphenhydramine (0.5-1.0 mg/kg; max dose 50 mg) are given i.v. over 10minutes to start 20 minutes prior to ch14.18/SP2/0 infusion;acetaminophen (10 mg/kg; max dose 650 mg) p.o. is given 20 minutes priorto ch14.18/SP2/0 infusion; and/or a morphine sulfate loading dose of 50mcg/kg is given immediately prior to ch14.18/SP2/0 administration andthen continued with morphine sulfate drip with an infusion rate of 20-50micrograms/kg/h to continue for two hours after completion of thech14.18/SP2/0 infusion. Additionally, other narcotics such ashydromorphone or fentanyl may be used. Alternatively, lidocaine infusionmay be used in conjunction with an i.v. bolus of morphine, if required.The usual administration guidelines for lidocaine infusion are shownbelow:

Administration of Lidocaine (in Usual Doses):

-   -   a. Give lidocaine i.v. bolus at 2 mg/kg in 50 cc normal saline        (NS) over 30 min prior to the start of ch14.18/SP2/0 infusion.    -   b. At the beginning of ch14.18/SP2/0 infusion, start i.v.        lidocaine infusion at 1 mg/kg/h and continue until two hours        after the completion of ch14.18/SP2/0 infusion.    -   c. May give morphine i.v. bolus 25-50 microgram/kg every 2 h, if        needed.

In said example of usual doses, one may also consider the administrationof gabapentin with loading doses of morphine, and give morphineinfusion/bolus as needed; may start with gabapentin 10 mg/kg/day andtitrate up to 30-60 mg/kg/day depending on the clinical response.

In said example of usual doses, doses of hydroxyzine (ordiphenhydramine) and acetaminophen can be repeated every 6 h, if needed;i.v. or p.o.

In said example of usual doses, additional morphine doses can be givenduring the ch14.18/SP2/0 infusion to treat neuropathic pain followed byan increase in the morphine sulfate infusion rate, but patients shouldbe monitored closely. If patients cannot tolerate morphine (e.g.,itching), fentanyl or hydromorphone can be substituted for morphine.Alternatively, lidocaine infusion may be used in conjunction with i.v.bolus of morphine, if needed.

The term “morphine dose” as used herein refers to the amount of morphine(in mg or mcg) per kg of body weight of the patient. Accordingly, if itis referred to a daily morphine dose, it is the amount of morphine (inmg or mcg) per kg of body weight of the patient per day, or if it isreferred to a morphine dose per hour, it is the amount of morphine (inmg or mcg) per kg of body weight of the patient per hour (or morphineinfusion rate), or if it is referred to a morphine dose per treatmentcycle, it is the amount of morphine (in mg or mcg) per kg of body weightof the patient per treatment cycle, or if it is referred to a morphinedose per overall treatment time, it is the amount of morphine (in mg ormcg) per kg of body weight of the patient per overall treatment time.

In some embodiments, the morphine and/or other analgesics may beadministered in a usual daily dose, but with a reduced frequency ofadministration, e.g. only every other day, if it has usually beenadministered each day. In another example, in which the morphine and/orother analgesics have usually been administered as continuous i.v.infusion over several days, either the infusion time per day may bereduced (resulting in a non-continuous infusion), or the total durationof the continuous infusion is reduced, i.e. the number of continuousinfusion days.

In some embodiments, the daily dose of the one or more analgesics on oneor more antibody treatment days (i.e. a day of a treatment cycle onwhich the preparation comprising an anti-GD2 antibody is administered tothe patient) in a treatment cycle according to the invention (with nocytokine treatment and/or in a continuous antibody infusion schedule) islower than the usual daily dose administered on one or more antibodytreatment days in a usual treatment cycle (with usual cytokine treatmentand/or in a non-continuous antibody infusion schedule). In someembodiments, the daily dose of the one or more analgesics on one or moreanalgesic treatment days (i.e. a day of a treatment cycle on which oneor more analgesics, e.g. morphine, is administered to the patient, whichmay also be days prior to or after antibody administration) in atreatment cycle according to the invention (with no cytokine(s)treatment and/or in a continuous antibody infusion schedule) is lowerthan the usual daily dose administered on one or more analgesictreatment days in a usual treatment cycle (with usual cytokine treatmentand/or in a non-continuous antibody infusion schedule).

In certain embodiments, the dose (e.g. the daily dose) of the one ormore analgesics (e.g. morphine) is reduced over time, e.g. within theoverall treatment time, within a treatment cycle, during the antibodytreatment period within a treatment cycle, from one antibody oranalgesic treatment day to the next antibody or analgesic treatment daywithin a treatment cycle, and/or from one treatment cycle to the next.In some embodiments, the analgesics dose, in particular the morphinedose, is continuously reduced within a treatment cycle, during theantibody treatment period within a treatment cycle, and/or from oneantibody or analgesic/morphine treatment day to the next antibody oranalgesic/morphine treatment day within a treatment cycle.

In some embodiments, the daily morphine dose administered during one ormore days of administration of the antibody in a continuous antibodyinfusion schedule and/or an antibody administration schedule with nocytokine according to the invention, and/or the morphine dose of allantibody or morphine treatment days is lower than the daily morphinedose during administration of the antibody with the usual doses of oneor more cytokines and/or in a non-continuous antibody administrationschedule. In certain embodiments, the daily morphine dose (and/or themorphine dose per treatment cycle) administered in an antibody infusionschedule according to the invention (e.g. with no cytokine and/or ascontinuous antibody infusion) is 90% or less, 80% or less, 70% or less,60% or less, or 50% or less, or 40% or less, or 30% or less, or 20% orless, or 10% or less of the morphine dose administered in or prescribedfor a usual antibody infusion schedule (e.g. with usual cytokine dosesand/or in a non-continuous antibody infusion). In some embodiments, thedaily morphine dose administered during one or more days of continuousintravenous infusion of the antibody with no cytokine according to theinvention and/or of all antibody or morphine treatment days is lowerthan 0.9, 0.72, 0.48, 0.38, 0.4375, and/or 0.205 mg/kg/day.

In some embodiments, the daily morphine dose administered during one ormore days of continuous intravenous infusion of the antibody accordingto the invention and/or of all morphine treatment days is lower than thedaily morphine dose during non-continuous administration of theantibody, e.g. in one or more of the examples described above. Incertain embodiments, the daily morphine dose administered in acontinuous antibody infusion schedule is 80% or less of the morphinedose administered in or prescribed for a non-continuous antibodyinfusion schedule in the first treatment cycle, 58% or less in thesecond treatment cycle, 57% or less in the third treatment cycle, 42% orless in the fourth treatment cycle, 34% or less in the fifth treatmentcycle. In one embodiment, the daily morphine dose administered in anantibody treatment schedule according to the invention (e.g. with nocytokine and/or as continuous antibody infusion) is lower than the dailymorphine dose administered in an antibody treatment schedule of acontinuous intravenous antibody infusion for 5 days in a daily dose of40 mg/m². In some embodiments, the daily morphine dose administeredduring one or more days of continuous intravenous infusion of theantibody according to the invention and/or of all antibody or morphinetreatment days is lower than 0.9, 0.72, 0.48, 0.38, 0.4375, and/or 0.205mg/kg/day.

In certain embodiments, the daily morphine dose administered on thefifth, sixth, seventh, eighths, ninth, and/or tenth day ofadministration of the antibody in the first treatment cycle (applying acontinuous intravenous infusion of the antibody according to theinvention) is lower than the daily morphine dose in a non-continuousinfusion schedule, e.g. 78% or less. In certain embodiments, themorphine dose administered on said day and any following days of thetreatment cycle and/or of the overall treatment time is lower than thedaily morphine dose in a non-continuous infusion schedule. In certainembodiments, the daily morphine dose administered on the third, fourth,fifth, sixth, seventh, eighths, ninth, and/or tenth day ofadministration of the antibody in the second treatment cycle (applying acontinuous intravenous infusion of the antibody according to theinvention) is lower than the daily morphine dose in a non-continuousinfusion schedule, e.g. 60% or less. In certain embodiments, themorphine dose administered on said day and any following days of thetreatment cycle and/or of the overall treatment time is lower than thedaily morphine dose in a non-continuous infusion schedule. In someembodiments, the daily morphine dose administered on the first day ofadministration of the antibody in the third and any following treatmentcycles (applying a continuous intravenous infusion of the antibodyaccording to the invention) is lower than the daily morphine dose in anon-continuous infusion schedule, e.g. 57% or less. In certainembodiments, the morphine dose administered on said day and anyfollowing days of the treatment cycle and/or of the overall treatmenttime is lower than the daily morphine dose in a non-continuous infusionschedule.

In some embodiments of inventive treatment schedules, morphine isadministered only for some but not all days on which the antibody isadministered, e.g. only on the first 1, 2, 3, 4, 5, 6, or 7 days (e.g.in treatment cycles two, three, four, and/or five) of continuousantibody infusion and/or of antibody administration with no cytokine. Insome embodiments, no morphine is administered on one or more days of theinventive antibody administration schedule, or even within the entiretreatment cycle. In some embodiments, morphine is administered only insome but not all treatment cycles. In an embodiment, in cycle 6 ofcontinuous antibody infusion, no morphine is administered.

In some embodiments, the morphine infusion rate, i.e. the morphineamount per kg body weight of the patient (or morphine dose) per hour,administered during one or more hours or days of the continuousintravenous infusion of the antibody according to the invention and/orthe antibody administration with no cytokine according to the inventionand/or of all hours or days of morphine treatment is lower than thestandard morphine infusion rate prescribed for a usual schedule withnon-continuous infusion and/or with usual cytokine doses, and/or lowerthan the morphine infusion rate in the examples with non-continuousinfusion and/or usual cytokine doses described above. In someembodiments, the morphine infusion rate, i.e. the morphine amount per kgbody weight of the patient (or morphine dose) per hour, administeredduring one or more hours or days of the continuous intravenous infusionof the antibody according to the invention and/or of all hours or daysof morphine treatment is lower than the standard morphine infusion rateprescribed for said usual schedule and/or the morphine infusion rateduring non-continuous administration of the antibody in the examplesdescribed above, e.g. 96% or less on the second, 84% or less on thethird, 65% or less on the fourth, 41% or less on the fifth, 14% on thesixth, 5% or less on the seventh, 3% on the eighth, 2% or less on theninth, and/or 1% on the tenth day in the first treatment cycle, 72% orless in the second treatment cycle, 30% or less in the third treatmentcycle, 22% or less in the fourth treatment cycle, 18% or less in thefifth treatment cycle. In some embodiments, the morphine infusion rateadministered during one or more days of intravenous infusion of theantibody with no cytokine according to the invention and/or of allantibody or morphine treatment days is lower than 50, 40, 30, 20, 10,and/or 5 mcg/kg/h, and/or lower than any range in between these infusionrates. In some embodiments, the morphine infusion rate administeredduring one or more days of continuous intravenous infusion of theantibody according to the invention and/or of all antibody or morphinetreatment days is lower than 50, 40, 30, 20, 10, and/or 5 mcg/kg/h,and/or lower than any range in between these infusion rates. In someembodiments, the morphine infusion rate administered during one or moredays of continuous intravenous infusion of the antibody according to theinvention and/or of all antibody or morphine treatment days is lowerthan 30 mcg/kg/h in the first and optionally any following treatmentcycles, lower than 22 mcg/kg/h in the second and optionally anyfollowing treatment cycles, lower than 10 mcg/kg/h in the third andoptionally any following treatment cycles, lower than 7 mcg/kg/h in thefourth and optionally any following treatment cycles, and/or lower than6 mcg/kg/h in the fifth and optionally any following treatment cycles.In some embodiments, the morphine infusion rate administered during oneor more days of intravenous infusion of the antibody with no cytokineaccording to the invention and/or of all antibody or morphine treatmentdays is lower than 50 mcg/kg/h in the first and optionally any followingtreatment cycles, lower than 40 mcg/kg/h in the second and optionallyany following treatment cycles, lower than 30 mcg/kg/h in the third andoptionally any following treatment cycles, lower than 20 mcg/kg/h in thefourth and optionally any following treatment cycles, and/or lower than10 mcg/kg/h in the fifth and optionally any following treatment cycles.

In certain embodiments, the morphine dose per treatment cycleadministered during one or more treatment cycles comprising thecontinuous intravenous infusion of the antibody according to theinvention is lower than the morphine dose per treatment cycle in anon-continuous infusion schedule, e.g. 66% or less in the firsttreatment cycle; 64% or less, or 28% or less in the second treatmentcycle; 29% or less, or 13% or less in the third treatment cycle; 16% orless, or 7% or less in the fourth treatment cycle; and/or 15% or less,or 6% or less in the fifth treatment cycle.

In certain embodiments, the morphine dose per treatment cycleadministered during one or more treatment cycles comprising theintravenous infusion of the antibody with no cytokine according to theinvention is lower than the morphine dose per treatment cycle in aninfusion schedule with usual cytokine treatment, e.g. 90%, 80%, 70%, orless in the first treatment cycle; 80%, 70%, 60%, or less in the secondtreatment cycle; 60%, 50%, 40%, or less in the third treatment cycle;45%, 35%, 25%, or less in the fourth treatment cycle; and/or 30%, 20%,10% or less in the fifth treatment cycle.

In certain embodiments, the morphine dose per treatment cycle of thesecond and any following treatment cycles administered during one ormore treatment cycles comprising the continuous intravenous infusion ofthe antibody and/or the antibody administration with no cytokineaccording to the invention is lower than the morphine dose per treatmentcycle in a non-continuous infusion schedule and/or in an administrationschedule of an antibody with usual cytokine doses. In certainembodiments, the morphine dose of said treatment cycle and any followingtreatment cycles, and/or of the overall treatment time is lower than themorphine dose per treatment cycle in a usual infusion schedule (e.g.non-continuous and/or with usual cytokine doses). In some embodiments,the morphine dose per treatment cycle administered during one or moretreatment cycles comprising the continuous intravenous infusion of theantibody and/or the antibody administration with no cytokine accordingto the invention is lower than 7.2, 4.8, 4.5, 2, 1.75, and/or 0.82mg/kg/cycle, or lower than any range in between these doses.

In some embodiments, the morphine dose of the overall treatment time inan inventive infusion schedule (e.g. applying a continuous intravenousinfusion of the antibody and/or applying an antibody administrationschedule with no cytokine(s)) is lower than the morphine dose of theoverall treatment time in a usual infusion schedule (e.g. an antibodyinfusion schedule with the usual cytokine dose(s) and/or anon-continuous antibody infusion schedule).

In one embodiment, the morphine dose of the overall treatment time(applying a continuous intravenous infusion of the antibody according tothe invention) is 55% or less, 50% or less, 45% or less, 40% or less,30% or less, 20% or less, 10% or less, of the morphine dose of theoverall treatment time in a non-continuous infusion schedule, and/orlower than any range in between these doses. In some embodiments, themorphine dose of the overall treatment time (applying a continuousintravenous infusion of the antibody according to the invention) islower than 43.2, 28.8, 13.5, 10, 8.75, and/or 4.1 mg/kg/overalltreatment time, and/or lower than any range in between these doses.

In one embodiment, the morphine dose of the overall treatment time in aninventive infusion schedule (with no cytokine(s) treatment and/or in acontinuous antibody infusion schedule) is 90% or less, 80% or less, 70%or less, 60% or less, or 50% or less, 40% or less, 30% or less, 20% orless, 10% or less, of the morphine dose of the overall treatment time ina usual infusion schedule (with usual cytokine(s) treatment and/or in anon-continuous antibody infusion schedule), and/or lower than any rangein between these doses. In some embodiments, the morphine dose of theoverall treatment time in an inventive infusion schedule withoutcytokine treatment and/or with continuous antibody infusion is lowerthan 43.2, 28.8, 13.5, 10, 8.75, and/or 4.1 mg/kg/overall treatmenttime, and/or lower than any range in between these doses.

In some embodiments, the reference morphine doses in usual infusionschedules (with usual cytokine(s) treatment schedules and/or in anon-continuous antibody infusion schedule), as referred to herein incomparison to the morphine doses in infusion schedules according to thepresent invention (with no cytokine(s) treatment and/or in a continuousantibody infusion schedule), refer to the standard morphine doses forsaid schedule, or morphine doses prescribed for said schedule (e.g. asspecified in the clinical study protocols). In some embodiments, thereference morphine doses as referred to herein refers to the morphinedose administered on the first day of treatment with the preparationcomprising an anti-GD2 antibody in a treatment cycle with an inventiveand/or usual antibody infusion schedule, and is referred to as “startingmorphine dose”.

Accordingly, the term “reference morphine dose” as used herein shallcomprise the morphine doses of treatment schedules other than thoseaccording to the present invention (e.g. treatment schedules describedin the prior art, e.g. with usual cytokine treatment and/ornon-continuous antibody infusion), and/or starting morphine doses (e.g.of treatment schedules with or without cytokine treatment and/or with orwithout continuous antibody infusion); and shall encompass all examplesof such morphine doses as referred to herein in comparison to the othermorphine doses in antibody infusion schedules with no cytokine(s) and/orwith continuous antibody infusion according to the present invention.

In certain embodiments, the reference morphine dose per hour infusion,i.e. the reference infusion rate, during the administration period ofthe antibody is 50 mcg/kg/h. In certain embodiments, the referencemorphine dose per hour infusion, i.e. the reference infusion rate,during the administration period of the antibody is 30 mcg/kg/h. Incertain embodiments, the reference morphine dose is 50, 40, 30, and/or20 mcg/kg/h. In certain embodiments, the reference morphine dose is 0.9,0.72, 0.48, 0.38, 0.4375, and/or 0.205 mg/kg/day. In certainembodiments, the reference morphine dose is 7.2, 4.8, 4.5, 2, 1.75,and/or 0.82 mg/kg/cycle. In certain embodiments, the reference morphinedose is 43.2, 28.8, 13.5, 10, 8.75, and/or 4.1 mg/kg/overall treatmenttime. In certain embodiments, the reference indometacin dose is 0.3 to0.5 mg/kg/dose, or 25 or 50 mg/dose, orally or intravenously every 6hours. In some embodiments, the reference morphine doses in usualinfusion schedules, as referred to above in comparison to the morphinedoses in inventive infusion schedules, refer to the morphine doses asactually administered to the patients (e.g. the respective mean of themorphine doses administered to all treated patients of a group treatedin the same setting).

The morphine doses in inventive infusion schedules, as referred toherein in comparison to the morphine doses in usual infusion schedules,may refer to the reduced standard morphine doses for said inventiveschedule, or reduced morphine doses prescribed for said schedule (e.g.as specified in the clinical study protocols). In certain embodiments,the reduced morphine dose in an inventive infusion schedule per hourinfusion, i.e. the infusion rate, during one or more hours or days ofthe inventive administration of the antibody with no cytokine(s) and/orin a continuous infusion is lower than 50 mcg/kg/h. In certainembodiments, the morphine dose per hour infusion, i.e. the infusionrate, during one or more hours or days of the inventive administrationof the antibody is lower than 30 mcg/kg/h. In some embodiments, themorphine doses in infusion schedules according to the present invention(e.g. without cytokine(s) and/or as continuous infusion), as referred toabove in comparison to the morphine doses in usual infusion schedules(e.g. with cytokine(s) and/or in non-continuous infusion), refer to themorphine doses as actually administered to the patients (e.g. therespective mean of the morphine doses administered to all treatedpatients of a group treated in the same setting).

In some embodiments, the reference morphine doses in non-continuousinfusion schedules, as referred to above in comparison to the morphinedoses in continuous infusion schedules according to the presentinvention, refer to the morphine doses as actually administered to thepatients (e.g. the respective mean of the morphine doses administered toall treated patients of a clinical study and/or in the same setting).

The morphine doses in continuous infusion schedules according to thepresent invention, as referred to herein in comparison to the morphinedoses in non-continuous infusion schedules, may refer to the standardmorphine doses for said schedule, or morphine doses prescribed for saidschedule (e.g. as specified in the clinical study protocols). In certainembodiments, the morphine dose per hour infusion, i.e. the infusionrate, during one or more hours or days of the continuous administrationof the antibody is lower than 50 mcg/kg/h. In certain embodiments, themorphine dose per hour infusion, i.e. the infusion rate, during one ormore hours or days of the continuous administration of the antibody islower than 30 mcg/kg/h.

In general, individual analgesic doses may vary depending on theindividual patient's pain tolerance. Dosing may be adapted to obtainoptimal analgesia.

The treatment period with the preparation comprising an anti-GD2antibody may be combined with one or more treatment periods with acytokine (according to the first aspect of the invention), one or moretreatment periods with a retinoid, one or more treatment periods with ananalgesic, and/or one or more treatment periods with another drug ortreatment (except for cytokine treatment in the second aspect of theinvention), and/or one or more treatment periods with no treatment. Inone embodiment, the treatment period with the preparation comprising ananti-GD2 antibody combined with one or more of any such other treatmentperiods represent one treatment cycle. Accordingly, in an embodiment, atreatment cycle comprises a treatment period with the preparationcomprising an anti-GD2 antibody and optionally one or more treatmentperiods with other drugs, agents, and/or treatments. Any such treatmentperiods within one treatment cycle may partially and/or entirelyoverlap, as further described herein.

In one embodiment, a patient who is treated with the antibody is alsotreated with a retinoid (e.g. isotretinoin), and optionally with one ormore cytokines (according to the first aspect of the invention), such asIL-2 and/or GM-CSF, and optionally morphine and/or one or more morphinederivatives, and/or one or more other analgesics. In one embodiment, thetreatment period with the preparation comprising an anti-GD2 antibody isnot accompanied by a treatment period with morphine and/or one or moreanalgesics. In one embodiment, the treatment period with the preparationcomprising an anti-GD2 antibody is not accompanied by a treatment periodwith one or more cytokines. In one embodiment, the treatment period withthe preparation comprising an anti-GD2 antibody is not preceded and/orfollowed by a treatment period with one or more cytokines.

In one embodiment, the treatment period with the preparation comprisingan anti-GD2 antibody is not preceded and/or followed by a treatmentperiod with one or more cytokines and not accompanied by a treatmentperiod with one or more cytokines.

In one embodiment, one treatment cycle comprises 28 to 49 days, e.g. 28,35, 42, or 49 days or any range in between these periods. The treatmentcycle starts with the day when the patient first receives any of thetreatments comprised in said cycle (may be designated as day 0 or day1), e.g. the administration of a preparation comprising an anti-GD2antibody, and/or any other preparation or treatment.

The treatment period with the anti-GD2 antibody may be preceded,accompanied, and/or followed by a treatment period with a retinoid (e.g.isotretinoin), either directly or with an interval of one or more daysof no treatment, e.g. 1, 2, 3, 4, or 5 days of no treatment. In oneembodiment, the retinoid (e.g. isotretinoin) is administered orallytwice a day in a dose of 160 mg/m²/day (in equal doses, i.e. 2×80mg/m²). In one embodiment, the retinoid (e.g. isotretinoin) isadministered for 14 days, e.g. from day 1 to day 14 of a treatmentcycle, or from day 19 to day 32 of the treatment cycle. The treatmentperiod with the retinoid (e.g. isotretinoin) may be followed by aninterval of one or more days of no treatment, e.g. 1, 2, 3, 4, or 5 daysof no treatment.

In one embodiment, the treatment cycle comprises one 14-day treatmentperiod with the retinoid (e.g. isotretinoin), e.g. on days 1 to 14 ofthe treatment cycle, followed by 7 days of no treatment (day 15-21 ofthe treatment cycle), and a 5-day treatment period with the anti-GD2antibody (e.g. with 20 mg/m²/day infused over 8 hours to administer adose of 100 mg/m²/cycle), e.g. on days 22 to 26 of the treatment cycle,before the next cycle begins on day 29, which is then day 1 of the nexttreatment cycle. In other embodiments, the treatment cycle comprises one14-day treatment period with a retinoid (e.g. isotretinoin), e.g. ondays 1 to 14 of the treatment cycle, followed by one 5-day treatmentperiod with the anti-GD2 antibody (e.g. with 20 mg/m²/day infused over 8hours to administer a dose of 100 mg/m²/cycle), e.g. on days 22 to 28 ofthe treatment cycle, before the next cycle begins on day 29, which isthen day 1 of the next treatment cycle.

In one embodiment, the treatment cycle comprises one 10-day treatmentperiod with the anti-GD2 antibody (e.g. with 10 or 15 mg/m²/day toadminister a dose of 100 or 150 mg/m²/cycle), e.g. on days 8 to 17 ofthe treatment cycle, and one 14-day treatment period with a retinoid(e.g. isotretinoin), e.g. on days 19 to 32 of the treatment cycle,followed by 3 days of no treatment, before the next cycle begins on day36, which is then day 1 of the second treatment cycle.

In one embodiment of the first aspect of the invention, the treatmentcycle comprises two 5-day treatment periods with the cytokine, e.g. ondays 1 to 5 and 8 to 12 of the treatment cycle, one 10-day treatmentperiod with the anti-GD2 antibody (e.g. with 10 or 15 mg/m²/day toadminister a dose of 100 or 150 mg/m²/cycle), e.g. on days 8 to 17 ofthe treatment cycle, and one 14-day treatment period with a retinoid(e.g. isotretinoin), e.g. on days 19 to 32 of the treatment cycle,followed by 3 days of no treatment, before the next cycle begins on day36, which is then day 1 of the second treatment cycle.

In one embodiment, the treatment cycle optionally comprises two 5-daytreatment periods with the cytokine, e.g. on days 1 to 5 and 8 to 12 ofthe treatment cycle, and comprises one 14-day treatment period with theanti-GD2 antibody (e.g. with 7 or 15 mg/m²/day to administer a dose of100 or 210 mg/m²/cycle), e.g. on days 8 to 21 of the treatment cycle,and further comprises one 14-day treatment period with a retinoid (e.g.isotretinoin), e.g. on days 26 to 39 of the treatment cycle, followed by3 days of no treatment, before the next cycle begins on day 43, which isthen day 1 of the second treatment cycle.

In one embodiment, the treatment cycle optionally comprises two 5-daytreatment periods with the cytokine, e.g. on days 1 to 5 and 8 to 12 ofthe treatment cycle, and comprises one 15-day treatment period with theanti-GD2 antibody (e.g. with 10 mg/m²/day to administer a dose of 150mg/m²/cycle), e.g. on days 8 to 22 of the treatment cycle, and one14-day treatment period with a retinoid (e.g. isotretinoin), e.g. ondays 26 to 39 of the treatment cycle, followed by 3 days of notreatment, before the next cycle begins on day 43, which is then day 1of the second treatment cycle.

In one embodiment, the treatment cycle optionally comprises two 5-daytreatment periods with the cytokine, e.g. on days 1 to 5 and 8 to 12 ofthe treatment cycle, and comprises one 21-day treatment period with theanti-GD2 antibody (e.g. with 7 or 10 mg/m²/day to administer a dose of150 or 210 mg/m²/cycle), e.g. on days 8 to 28 of the treatment cycle,and one 14-day treatment period with a retinoid (e.g. isotretinoin),e.g. on days 33 to 46 of the treatment cycle, followed by 3 days of notreatment, before the next cycle begins on day 50, which is then day 1of the second treatment cycle.

In one embodiment of the first aspect of the invention, the treatmentcycle comprises one 3-day treatment period with the immunocytokine (e.g.APN301), e.g. on days 4 to 6 of the treatment cycle, two treatmentperiods with the cytokine, e.g. GM-CSF, e.g. on days 1 and 2 and on 8 to14 of the treatment cycle, and one 14-day treatment period withisotretinoin, e.g. on days 11 to 24 of the treatment cycle, followed by4 days of no treatment, before the next cycle begins on day 29, which isthen day 1 of the second treatment cycle.

In one embodiment, the treatment cycle comprises one 4-day treatmentperiod with the preparation comprising the anti-GD2 antibody (e.g.ch14.18/SP2/0), for example administered in a dose of 25 mg/m²/day, e.g.on days 3 to 6 of a 24-day treatment cycle beginning with day 0, or ondays 7 to 10 of a 32-day treatment cycle beginning with day 0, e.g. for5 cycles; and one treatment period with RA, e.g. isotretinoin on days 10to 23 of a treatment cycle beginning with day 0, or on days 14 to 27 ofa treatment cycle beginning with day 0. In one embodiment of the firstaspect of the invention, the treatment cycle comprises one 4-daytreatment period with the preparation comprising the anti-GD2 antibody(e.g. ch14.18/SP2/0), for example administered in a dose of 25mg/m²/day, e.g. on days 3 to 6 of a 24-day treatment cycle beginningwith day 0 (if GM-CSF is used as cytokine), or e.g. on days 7 to 10 of a32-day treatment cycle beginning with day 0 (if IL-2 is used ascytokine), for 5 cycles (e.g. the first with GM-CSF, the second withIL-2, the third with GM-CSF, the fourth with IL-2, and the fifth withGM-CSF); one or two treatment periods with a cytokine (e.g. GM-CSF at250 micrograms/m²/day for 14 days, on days 0 to 13 of a treatment cyclebeginning with day 0; or Aldesleukin (IL-2) in a dose of 3 MIU/m²/day ondays 0 to 3 and 4.5 MIU/m²/day on days 7 to 10 of a treatment cyclebeginning with day 0, and one treatment period with RA, e.g.isotretinoin on days 10 to 23 of a treatment cycle beginning with day 0(if GM-CSF is used as cytokine), or on days 14 to 27 of a treatmentcycle beginning with day 0 (if IL-2 is used as cytokine). In anembodiment, the treatment schedule is as specified in Table 2, 3 and/or4 (but with continuous antibody infusion and/or without cytokine(s)).

The treatment cycle may be repeated, either identically or in an amendedform, e.g. with a different dose or schedule, or with differentadditional treatments (e.g. with one or more other analgesics and/orcytokines). Thus, the overall treatment time (e.g. the time periodcomprising all subsequent treatment cycles, or the overall continuoustreatment period) may comprise at least 1, or 2 or more cycles, or 10 ormore cycles. In one embodiment, the preparation comprising an anti-GD2antibody is administered to the patient in two or more treatment cycles.In an embodiment, the preparation comprising an anti-GD2 antibody isadministered to the patient as a continuous infusion in two or moretreatment cycles. In an embodiment, the preparation comprising ananti-GD2 antibody is administered to the patient as a continuousinfusion in two or more treatment cycles without concomitantlyadministering IL-2. In one embodiment, the overall treatment timecomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 cycles, or more. The overall treatment time may even compriseup to 20 or more treatment cycles. As described above, treatment cyclesmay comprise time periods of no treatment (intervals in which notreatment is administered to the patient, i.e. no antibody, no cytokine,no other drug). Thus, as used herein, the overall treatment time mayalso comprise said intervals of no treatment within treatment cycles.

In one embodiment, the 35, 42, or 49 day treatment cycle as specifiedabove is repeated at least 4 or 5 times, so that the overall continuoustreatment period comprises at least 5 or 6 treatment cycles.

The effect of the antibody treatment (e.g. as continuous infusion and/orwithout one or more cytokines) may be determined by a complementdependent cytolysis (CDC) assay or a whole blood test (WBT). The WBT isan assay in which the target cells or target components (i.e. cells,liposomes or other cell-like compartments to be lysed) are contactedwith appropriately anti-coagulated whole blood from the patient. The CDCassay can be, for example, a standard CDC assay as known in the art(e.g. as described in Indusogie et al., J Immunol 2000; 164 (8):4178-84;Zeng et al. Molecular Immunology 2005; 42 (11):1311-9; or inWO2005/070967). The CDC assay and/or the WBT may be done with GD2positive target cells, such as tumor cell lines of the GD2 positivecancer to be treated. For example, if the patient to be treated suffersfrom neuroblastoma, the cell line may be a neuroblastoma cell line, suchas e.g. LAN-1 human neuroblastoma cells. In another example, if thepatient to be treated suffers from melanoma, the cell line may be amelanoma cell line, such as e.g. M21 human melanoma cells. In stillanother example, the target cells of the CDC assay and/or the WBT aretumor cells obtained from the patient, i.e. autologous tumor cells ofthe patient. In another embodiment, the target component of the CDCassay and/or WBT is a liposome displaying GD2 on the surface. The targetcells or target components are labeled with a signaling component, e.g.with a radioactive component, such as ⁵¹Cr, or with a fluorescentcomponent, such as calcein. The signaling component is comprised by thetarget cell or target component, i.e. is inside of the target cell ortarget component (e.g. a liposome packed with the signaling componentand displaying GD2 on the surface), and is released upon lysis of thetarget cell or target component. Thus, the signaling component providesthe assay readout. The target cells or components loaded with thesignaling compound are contacted with the whole blood, serum, or plasmain a certain ratio. The whole blood, plasma, or serum may be diluted forthe CDC or WBT, e.g. in a ratio of 1:2 or higher, e.g. 1:4, 1:5, or1:10, or any range in between these ratios prior to adding it to thesample. However, it may also be added to the sample un-diluted. Thefinal concentration of the whole blood, plasma, or serum in the CDC orWBT sample may e.g. be in the range of 10 to 50%. Target cell or targetcomponent lysis can be measured by release of said signaling componentby a scintillation counter or spectrophotometry. For example, the targetcell or target component lysis can be measured by determining the amountof ⁵¹Cr released into the supernatant by a scintillation counter. Thepercentage of lysis may be determined by the following equation:100×(experimental release−spontaneous release)/(maximumrelease−spontaneous release).

For the CDC assay, the cytolytic components (or effector components) areprovided by serum or appropriately anti-coagulated plasma obtained fromthe patient or donor comprising the complement system components. Forthe WBT, the cytolytic components (or effector components) are providedby appropriately anti-coagulated whole blood obtained from the patientor donor comprising the complement system components as well as allcellular components, and also any further components comprised in wholeblood which might be relevant to the target cell lysis, as well as theinterplay of all components (e.g. complement activation is known toactivate certain effector cells such as granulocytes) For the CDC and/orWBT, the serum, plasma, or whole blood may be added to the target cellsor target components in different dilutions.

Furthermore, one or more samples of the CDC assay and/or WBT may bespiked with an anti-GD2 antibody in different concentrations, e.g. forgeneration of a standard curve.

In another embodiment, one or more anti-idiotypic (anti-id) anti-GD2antibodies recognizing the variable domain of anti-GD2 antibodies may beadded to a sample to inhibit the target cell lysis mediated by theantibody, e.g. as a negative control or to prove specificity of theassay and that the target cell lysis measured without the anti-idantibody is antibody-mediated or antibody dependent.

In certain embodiments, the increased level of cytolysis after the oneor more initial antibody treatment days (compared to the level ofcytolysis prior to antibody treatment) is maintained over the entiretreatment cycle. In some embodiments, said increased level of cytolysisis maintained over the overall treatment time, i.e. even for timeperiods, where the patient is not treated with the preparationcomprising an anti-GD2 antibody, i.e. in the intervals between thetreatment periods with the preparation comprising an anti-GD2 antibody(if any, i.e. if the patient is not treated continuously over theoverall treatment time with the preparation comprising an anti-GD2antibody). In one embodiment, the level of cytolysis (level of targetcell lysis, e.g. measured in a CDC assay or WBT) of a blood sample of apatient treated according to the present invention is increased comparedto the level of cytolysis prior to the first treatment period with thepreparation comprising the antibody over the entire time period startingfrom the end of the first antibody treatment period to the end of thelast treatment cycle, i.e. even between the time periods of antibodytreatment. In one embodiment, said level of cytolysis is at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or any range in betweenthese levels, over the entire time period starting from the end of thefirst antibody treatment period to the end of the last treatment cycle.In an embodiment, the level of cytolysis after the first, second, third,fourth, fifth, sixth, seventh, eights, ninths, and/or tenth treatmentperiod with the anti-GD2 antibody is at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or 100%, or any range in between these levels. In anembodiment, the level of cytolysis prior to the second, third, fourth,fifth, sixth, seventh, eights, ninths, and/or tenth treatment periodwith the anti-GD2 antibody is at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100%, or any range in between these levels.

DEFINITIONS OF SPECIFIC EMBODIMENTS

The present invention may be more specifically defined as follows:

Definition 1: A method for treating a GD2 positive cancer byadministering a preparation comprising an anti-GD2 antibody to a patientas a continuous intravenous infusion over 24 hours per day.

Definition 2: A method according to definition 1, wherein thepreparation comprising an anti-GD2 antibody is administered in a dosesufficient to induce tumor cell lysis (cytolysis threshold dose).

Definition 3: A method according to definition 2, wherein the cytolysisthreshold dose is determined individually for each patient.

Definition 4: A method according to definitions 2 or 3, wherein thecytolysis threshold dose is determined by a complement dependentcytolysis assay.

Definition 5: A method according to definitions 2 or 3, wherein thecytolysis threshold dose is determined by a whole blood test.

Definition 6: A method according to any of definitions 1 to 5, whereinthe cytolysis threshold dose is the dose determined in a specific CDCassay or WBT to induce at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or 100% of the maximal possibletarget cell lysis in that respective assay.

Definition 7: A method according to any of definitions 1 to 6, whereinthe cytolysis threshold dose is the dose determined in a specific CDCassay or WBT to induce 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% of the maximal possible target cell lysis inthat respective assay.

Definition 8: A method according to any of definitions 1 to 7, whereinthe cytolysis threshold dose is 470 to 1000, 470 to 10000, 1410 to 3000,or 2350 to 5000 ng/mL serum or plasma.

Definition 9: A method according to any of definitions 1 to 8, whereinthe cytolysis threshold dose is 2 to 250, 2 to 2500, 2 to 100, 5 to 200,6 to 750, 6 to 7500, 10 to 1250, 10 to 12500, 6 to 300, 10 to 500, 15 to600, or 25 to 1000 ng/mL whole blood.

Definition 10: A method according to any of definitions 1 to 9, whereinthe preparation comprising an anti-GD2 antibody is administered in adose to achieve the cytolysis threshold dose in the patient's serum,plasma or whole blood within 1, 2, 3, or 4 days of the treatment withthe preparation comprising an anti-GD2 antibody.

Definition 11: A method according to any of definitions 1 to 10, whereinthe threshold cytolysis is maintained even for one or more time periodswithin the overall treatment time, where the patient is not treated withthe preparation comprising an anti-GD2 antibody.

Definition 12: A method according to any of definitions 1 to 11, whereinthe level of cytolysis is maintained over the entire treatment cycle.

Definition 13: A method according to any of definitions 1 to 12, whereinthe level of cytolysis is maintained over the overall treatment time.

Definition 14: A method according to any of definitions 1 to 13, whereinthe preparation comprising an anti-GD2 antibody is administered in adaily dose of 1 to 30 mg/m², 1 to 35 mg/m², 1 to 50 mg/m², or 1 to 60mg/m².

Definition 15: A method according to any of definitions 1 to 14, whereinthe preparation comprising an anti-GD2 antibody is administered in adaily dose of 1, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 12, 15, 20, 25, 30,32, 35, 40, 45, 50, or 60 mg/m².

Definition 16: A method according to any of definitions 1 to 15, whereinthe preparation comprising an anti-GD2 antibody is administered for atreatment period until the predetermined overall patient dose has beenadministered.

Definition 17: A method according to any of definitions 1 to 16, whereinthe preparation comprising an anti-GD2 antibody is administered for atreatment period until a certain therapeutic effect has been reached.

Definition 18: A method according to any of definitions 1 to 17, whereinthe preparation comprising an anti-GD2 antibody is administered by usinga mini-pump.

Definition 19: A method according to any of definitions 1 to 18, whereinthe anti-GD2 antibody is a chimeric or humanized antibody.

Definition 20: A method according to any of definitions 1 to 19, whereinthe anti-GD2 antibody is ch14.18/CHO or ch14.18/SP2/0.

Definition 21: A method according to any of definitions 1 to 20, whereinthe preparation comprising the anti-GD2 antibody is APN311 or APN301.

Definition 22: A method according to any of definitions 1 to 21, whereinthe preparation comprising an anti-GD2 antibody is administered in adose of 7, 10, 15, or 25 mg/m²/day.

Definition 23: A method according to any of definitions 1 to 22, whereinthe preparation comprising an anti-GD2 antibody is administered for 4,10, 14, 15, or 21 consecutive days.

Definition 24: A method according to any of definitions 1 to 23, whereinthe preparation comprising an anti-GD2 antibody is administered for 3,4, 5, or 6 treatment cycles.

Definition 25: A method according to any of definitions 1 to 24, whereinthe preparation comprising an anti-GD2 antibody is APN311 and isadministered in a dose of 10 mg/m²/day for 10 consecutive days for 6treatment cycles.

Definition 26: A method according to any of definitions 1 to 24, whereinthe anti-GD2 antibody is ch14.18/SP2/0 and is administered in a dose of25 mg/m²/day for 4 consecutive days for 5 treatment cycles.

Definition 27: A method according to any of definitions 1 to 26, whereinthe administration of the preparation comprising an anti-GD2 antibody ispreceded and/or accompanied by the administration of IL-2 and/or GM-CSFor another cytokine.

Definition 28: A method according to any of definitions 1 to 27, whereinthe administration period of the preparation comprising an anti-GD2antibody may be followed by an administration period of isotretinoin oranother retinoid.

Definition 29: A method according to any of definitions 1 to 28, whereinthe administration of the preparation comprising an anti-GD2 antibody isaccompanied by the administration of morphine and/or one or more otheranalgesics.

Definition 30: A method according to any of definitions 1 to 29, whereinthe daily morphine dose administered during one or more days ofcontinuous intravenous infusion of the antibody according to theinvention and/or of all morphine treatment days is lower than the dailymorphine dose during non-continuous administration of the antibody.

Definition 31: A method according to any of definitions 1 to 30, whereinmorphine is administered only for some but not all days on which theantibody is administered.

Definition 32: A method according to any of definitions 1 to 31, whereinthe morphine dose per treatment cycle administered during one or moretreatment cycles comprising the continuous intravenous infusion of theantibody according to the invention is lower than the morphine dose pertreatment cycle in a non-continuous infusion schedule.

Definition 33: A method according to any of definitions 1 to 32, whereinthe morphine dose of the overall treatment time is lower than themorphine dose of the overall treatment time in a non-continuous infusionschedule.

Definition 34: A method according to any of definitions 1 to 33, whereinthe morphine dose administered during one or more hours or days ofcontinuous intravenous infusion of the antibody according to theinvention and/or of all morphine treatment hours or days is lower than50 mcg/kg/h, or lower than 30 mcg/kg/h.

Definition 35: A method according to any of definitions 1 to 34, whereinthe daily morphine dose administered during one or more days ofcontinuous intravenous infusion of the antibody according to theinvention and/or of all morphine treatment days is lower than 0.9, 0.72,0.48, 0.38, 0.4375, and/or 0.205 mg/kg/day.

Definition 36: A method according to any of definitions 1 to 35, whereinthe dose of the one or more analgesics, especially morphine, is reducedwithin the overall treatment time, within a treatment cycle, during theantibody treatment period within a treatment cycle, from one antibodytreatment day to the next antibody treatment day within a treatmentcycle, and/or from one treatment cycle to the next.

Definition 37: A method according to any of definitions 1 to 36, whereinthe morphine dose is continuously reduced within a treatment cycle,during the antibody treatment period within a treatment cycle, and/orfrom one antibody treatment day to the next antibody treatment daywithin a treatment cycle.

Definition 38: An anti-GD2 antibody for use in a treatment according toany one of method definitions 1 to 37.

Definition 39: Use of an anti-GD2 antibody in the preparation of amedicament for the treatment according to any one of method definitions1 to 37.

Definition 40: A preparation comprising an anti-GD2 antibody for use inthe treatment of a GD2 positive cancer in a patient, wherein thepreparation comprising an anti-GD2 antibody is administered to thepatient without concomitantly administering IL-2, and wherein one ormore treatment periods with the antibody is/are preceded, accompanied,and/or followed by one or more treatment periods with a retinoid.

Definition 41: A preparation comprising an anti-GD2 antibody for use inthe treatment of a GD2 positive cancer in a patient, wherein thepreparation comprising an anti-GD2 antibody is administered to thepatient as a continuous infusion for one or more days and for two ormore treatment cycles, without concomitantly administering IL-2.

Definition 42: A preparation comprising an anti-GD2 antibody for use inthe treatment of a GD2 positive cancer in a patient, wherein thepreparation comprising an anti-GD2 antibody is administered to thepatient as a continuous infusion without concomitantly administeringIL-2, and wherein the anti-GD2 antibody is not a 14G2a antibody.

Definition 43: A method for treating a GD2 positive cancer byadministering a preparation comprising an anti-GD2 antibody to apatient, wherein the patient is not concomitantly treated with IL-2, andwherein one or more treatment periods with the antibody is/are preceded,accompanied, and/or followed by one or more treatment periods with aretinoid.

Definition 44: A method for treating a GD2 positive cancer in a patient,wherein a preparation comprising an anti-GD2 antibody is administered tothe patient as a continuous infusion for one or more days and for two ormore treatment cycles, and wherein the patient is not concomitantlytreated with IL-2.

Definition 45: A method for treating a GD2 positive cancer in a patient,wherein the preparation comprising an anti-GD2 antibody is administeredto the patient as a continuous infusion, wherein the patient is notconcomitantly treated with IL-2, and wherein the anti-GD2 antibody isnot a 14G2a antibody.

Definition 46: The preparation or method of any of definitions 40-45,wherein the patient is not concomitantly treated with GM-CSF.

Definition 47: The preparation or method of any of definitions 40-46,wherein the patient is not treated with a cytokine.

Definition 48: The preparation or method of any of definitions 40-47,wherein the patient is not treated with said cytokine(s) within the sametreatment cycle.

Definition 49: The preparation or method of any of definitions 40-48,wherein the patient is not treated with said cytokine(s) within the sameoverall treatment period.

Definition 50: The preparation or method of any of definitions 40-49,wherein the patient may have been treated with IL-2, GM-CSF, and/or oneor more other cytokines in one or more previous treatment cycles and/oroverall treatment periods.

Definition 51: The preparation or method of any of definitions 40-50,wherein the GD2 positive cancer is neuroblastoma.

Definition 52: The preparation or method of any of definitions 40-51,wherein the patient has been diagnosed with minimal residual disease.

Definition 53: The preparation or method of any of definitions 40-52,wherein the patient has been diagnosed with relapsed and/or refractorydisease.

Definition 54: The preparation or method of any of definitions 40-53,wherein the preparation comprising an anti-GD2 antibody is administeredas a continuous intravenous infusion over 24 hours per day.

Definition 55: The preparation or method of any of definitions 40-54,wherein the preparation comprising an anti-GD2 antibody is administeredin a daily dose of 1 to 30 mg/m², 1 to 35 mg/m², 1 to 50 mg/m², or 1 to60 mg/m².

Definition 56: The preparation or method of any of definitions 40-55,wherein the preparation comprising an anti-GD2 antibody is administeredby using a mini-pump.

Definition 57: The preparation or method of any of definitions 40-56,wherein the anti-GD2 antibody is ch14.18/CHO or ch14.18/SP2/0.

Definition 58: The preparation or method of any of definitions 40-57,wherein the preparation comprising the anti-GD2 antibody is APN311.

Definition 59: The preparation or method of any of definitions 40-58,wherein the preparation comprising an anti-GD2 antibody is administeredin a dose of 7, 10, 15, or 25 mg/m²/day.

Definition 60: The preparation or method of any of definitions 40-59,wherein the preparation comprising an anti-GD2 antibody is administeredfor 4, 10, 14, 15, or 21 consecutive days.

Definition 61: The preparation or method of any of definitions 40-60,wherein the preparation comprising an anti-GD2 antibody is administeredfor 3, 4, 5, 6, 7, 8, or 9 or more treatment cycles.

Definition 62: The preparation or method of any of definitions 40-61,wherein the preparation comprising an anti-GD2 antibody is APN311 and isadministered in a dose of 10 mg/m²/day for 10 consecutive days for 6 ormore treatment cycles.

Definition 63: The preparation or method of any of definitions 40-62,wherein the anti-GD2 antibody is ch14.18/SP2/0 and is administered in adose of 25 mg/m²/day for 4 consecutive days for 5 or more treatmentcycles.

Definition 64: The preparation or method of any of definitions 40-63,wherein the administration period of the preparation comprising ananti-GD2 antibody may be followed by an administration period ofisotretinoin or another retinoid.

Definition 65: The preparation or method of any of definitions 40-64,wherein the administration of the preparation comprising an anti-GD2antibody is accompanied by the administration of morphine and/or one ormore other analgesics.

Definition 66: The preparation or method of any of definitions 40-65,wherein the administration of the preparation comprising an anti-GD2antibody is accompanied by the administration of a reduced dose ofmorphine and/or one or more other analgesics.

Definition 67: The preparation or method of any of definitions 40-66,wherein the administration of the preparation comprising an anti-GD2antibody is not accompanied by the administration of morphine and/or oneor more other analgesics.

EXAMPLES Example 1: APN311 and APN301 Sequences and Related Data

APN311 Sequence Data

TABLE 5 Molecular Weight (MW) and pI (calculated) MW No. 2D- pI ¹⁾ [D]¹⁾ of AS Conditions DIGE ²⁾ Antibody 8.61 144701.10 1324 non-reducing xAntibody (½) 8.58 72359.56 662 reducing x Heavy Chain 8.58 48306.59 442reducing x Light Chain 8.48 24070.98 220 reducing x ¹⁾ Calculated viahttp://web.expasy.org/compute_pi/ ²⁾ Due to the molecular weight of thedyes, shifts to slightly higher molecular weights are to be expected for2D-DIGENucleotide Sequence (cDNA, Incl. Leader)

“TAG” works as a “stop codon” and therefore is not translated into thepeptide sequence.

Light Chain (SEQ ID NO: 1): 1

61GAA ATA GTG ATG ACG CAG TCT CCA GCC ACC CTG TCT GTG TCT CCA GGG GAA AGA GCC ACC121CTC TCC TGC AGA TCT AGT CAG AGT CTT GTA CAC CGT AAT GGA AAC ACC TAT TTA CAT TGG181TAC CTG CAG AAG CCA GGC CAG TCT CCA AAG CTC CTG ATT CAC AAA GTT TCC AAC CGA TTT241TCT GGG GTC CCA GAC AGG TTC AGT GGC AGT GGA TCA GGG ACA GAT TTC ACA CTC AAG ATC301AGC AGA GTG GAG GCT GAG GAT CTG GGA GTT TAT TTC TGT TCT CAA AGT ACA CAT GTT CCT361CCG CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA CGA ACT GTG GCT GCA CCA TCT421GTC TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG AAA TCT GGA ACT GCC TCT GTT GTG TGC481CTG CTG AAT AAC TTC TAT CCC AGA GAG GCC AAA GTA CAG TGG AAG GTG GAT AAC GCC CTC541CAA TCG GGT AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC TAC AGC601CTC AGC AGC ACC CTG ACG CTG AGC AAA GCA GAC TAC GAG AAA CAC AAA GTC TAC GCC TGC661GAA GTC ACC CAT CAG GGC CTG AGC TCG CCC GTC ACA AAG AGC TTC AAC AGG GGA GAG TGT721

Heavy Chain (SEQ ID NO: 2): 1

61GTC CAA CTG CTG CAG TCT GGA CCT GAG CTG GAG AAG CCT GGC GCT TCA GTG ATG ATA TCC121TGC AAG GCT TCT GGT TCC TCA TTC ACT GGC TAC AAC ATG AAC TGG GTG AGG CAG AAC ATT181GGA AAG AGC CTT GAA TGG ATT GGA GCT ATT GAT CCT TAC TAT GGT GGA ACT AGC TAC AAC241CAG AAG TTC AAG GGC AGG GCC ACA TTG ACT GTA GAC AAA TCG TCC AGC ACA GCC TAC ATG301CAC CTC AAG AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TAC TGT GTA AGC GGA ATG GAG361TAC TGG GGT CAA GGA ACC TCA GTC ACC GTC TCC TCA GCC TCC ACC AAG GGC CCA TCG GTC421TTC CCC CTG GCA CCC TCC TCC AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GGC TGC CTG481GTC AAG GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC CTG ACC AGC541GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TAC TCC CTC AGC AGC GTG601GTG ACC GTG CCC TCC AGC AGC TTG GGC ACC CAG ACC TAC ATC TGC AAC GTG AAT CAC AAG661CCC AGC AAC ACC AAG GTG GAC AAG AGA GTT GAG CCC AAA TCT TGT GAC AAA ACT CAC ACA721TGC CCA CCG TGC CCA GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA781AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC841GTG AGC CAC GAA GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT901AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC GTC961CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC1021AAA GCC CTC CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA1081CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG1141ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG1201CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC1261CTC TAT AGC AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC1321TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCC CCG1381

nucleotide 1 to 60 (striked out): leader sequence last nucleotide(striked out): stop codonPeptide Sequence (Incl. Signal Peptide)

The signal peptide is split off during post translational processing andis not part of the final recombinant protein anymore.

Light Chain (SEQ ID NO: 3): 1

21 E I V M T Q S P A T L S V S P G E R A T 41L S C R S S Q S L V H R N G N T Y L H W 61Y L Q K P G Q S P K L L I H K V S N R F 81S G V P D R F S G S G S G T D F T L K I 101S R V E A E D L G V Y F C S Q S T H V P 121P L T F G A G T K L E L K R T V A A P S 141 V F I F P P S D E Q L K S G T A S V V C 161 L L N N F Y P R E A K V Q W K V D N A L 181Q S G N S Q E S V T E Q D S K D S T Y S 201L S S T L T L S K A D Y E K H K V Y A C 221E V T H Q G L S S P V T K S F N R G E C Heavy Chain (SEQ ID NO: 4): 1

21 V Q L L Q S G P E L E K P G A S V M I S 41C K A S G S S F T G Y N M N W V R Q N I 61G K S L E W I G A I D P Y Y G G T S Y N 81Q K F K G R A T L T V D K S S S T A Y M 101H L K S L T S E D S A V Y Y C V S G M E 121Y W G Q G T S V T V S S A S T K G P S V 141F P L A P S S K S T S G G T A A L G C L 161V K D Y F P E P V T V S W N S G A L T S 181G V H T F P A V L Q S S G L Y S L S S V 201V T V P S S S L G T Q T Y I C N V N H K 221P S N T K V D K R V E P K S C D K T H T 241C P P C P A P E L L G G P S V F L F P P 261K P K D T L M I S R T P E V T C V V V D 281V S H E D P E V K F N W Y V D G V E V H 301N A K T K P R E E Q Y N S T Y R V V S V 321L T V L H Q D W L N G K E Y K C K V S N 341K A L P A P I E K T I S K A K G Q P R E 361P Q V Y T L P P S R E E M T K N Q V S L 381T C L V K G F Y P S D I A V E W E S N G 401Q P E N N Y K T T P P V L D S D G S F F 421L Y S K L T V D K S R W Q Q G N V F S C 441S V M H E A L H N H Y T Q K S L S L S P 461 G K amino acid 1 to 20(striked out): leader sequenceAPN301 Sequence Data

TABLE 6 Molecular Weight (MW) and pI (calculated) MW No. 2D- pI ¹⁾ [D]¹⁾ of AS Conditions DIGE ²⁾ Immunocytokine 8.52 175741.35 1592non-reducing x Antibody 8.61 144941.37 1326 non-reducing Immunocytokine8.49 87879.68 796 reducing x (½) Antibody (½) 8.57 72479.69 663 reducingHeavy Chain + IL-2 8.47 63861.72 576 reducing x Heavy Chain 8.5948461.73 443 reducing Light Chain 8.27 24035.97 220 reducing x IL-2 7.0515418.01 133 reducing ¹⁾ Calculated viahttp://web.expasy.org/compute_pi/ ²⁾ Due to the molecular weight of thedyes, shifts to slightly higher molecular weights are to be expected for2D-DIGE 3) IL-2 should not be cleaved off the immunocytokine underreducing condition, as it is bound covalently via a linker to the Fcportion and therefore the heavy chain, antibody (½) and the antibodyshould not be present on a 2D-DIGENucleotide Sequence (cDNA, Incl. Leader)

“TAG” and “TGA” work as “stop codons” and therefore are not translatedinto the peptide sequence.

Light Chain (SEQ ID NO: 5): 1

61GTG GTG ATG ACC CAG ACC CCC CTG TCC CTG CCC GTG ACC CCC GGC GAG CCC GCC TCC ATC121TCC TGC AGA TCT AGT CAG AGT CTT GTA CAC CGT AAT GGA AAC ACC TAT TTA CAT TGG TAC181CTG CAG AAG CCA GGC CAG TCT CCA AAG CTC CTG ATT CAC AAA GTT TCC AAC CGA TTT TCT241GGG GTC CCA GAC AGG TTC AGT GGC AGT GGA TCA GGG ACA GAT TTC ACA CTC AAG ATC AGC301AGA GTG GAG GCT GAG GAT CTG GGA GTT TAT TTC TGT TCT CAA AGT ACA CAT GTT CCT CCG361CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA CGA ACT GTG GCT GCA CCA TCT GTC421TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG AAA TCT GGA ACT GCC TCT GTT GTG TGC CTG481CTG AAT AAC TTC TAT CCC AGA GAG GCC AAA GTA CAG TGG AAG GTG GAT AAC GCC CTC CAA541TCG GGT AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC TAC AGC CTC601AGC AGC ACC CTG ACG CTG AGC AAA GCA GAC TAC GAG AAA CAC AAA GTC TAC GCC TGC GAA661

Heavy Chain  (incl. IL-2; SEQ ID NO: 6:) 1

61GTG CAG CTG GTG CAG TCC GGC GCC GAG GTG GAG AAG CCC GGC GCC TCC GTG AAG ATC TCC121TGC AAG GCC TCC GGC TCC TCC TTC ACC GGC TAC AAC ATG AAC TGG GTG CGC CAG AAC ATC181GGC AAG TCC CTG GAG TGG ATC GGC GCC ATC GAC CCC TAC TAC GGC GGC ACC TCC TAC AAC241CAG AAG TTC AAG GGC CGC GCC ACC CTG ACC GTG GAC AAG TCC ACC TCC ACC GCC TAC ATG301CAC CTG AAG TCC CTG CGC TCC GAG GAC ACC GCC GTG TAC TAC TGC GTG TCC GGC ATG GAG361TAC TGG GGC CAG GGC ACC TCC GTG ACC GTG TCC TCC GCC TCC ACC AAG GGC CCA TCG GTC421TTC CCC CTG GCA CCC TCC TCC AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GGC TGC CTG481GTC AAG GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC CTG ACC AGC541GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TAC TCC CTC AGC AGC GTG601GTG ACC GTG CCC TCC AGC AGC TTG GGC ACC CAG ACC TAC ATC TGC AAC GTG AAT CAC AAG661CCC AGC AAC ACC AAG GTG GAC AAG AGA GTT GAG CCC AAA TCT TGT GAC AAA ACT CAC ACA721TGC CCA CCG TGC CCA GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA781AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC841GTG AGC CAC GAA GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT901AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC GTC961CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC1021AAA GCC CTC CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA1081CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG1141ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG1201CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC1261CTC TAT AGC AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC1321TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCC CCG1381GGT AAA GCC CCA ACT TCA AGT TCT ACA AAG AAA ACA CAG CTG CAA CTG GAG CAT CTC CTG1441CTG GAT CTC CAG ATG ATT CTG AAT GGA ATT AAC AAC TAC AAG AAT CCC AAA CTC ACC AGG1501ATG CTC ACA TTC AAG TTC TAC ATG CCC AAG AAG GCC ACA GAG CTC AAA CAT CTC CAG TGT1561CTA GAG GAG GAA CTC AAA CCT CTG GAG GAA GTG CTA AAC CTC GCT CAG AGC AAA AAC TTC1621CAC TTA AGA CCT AGG GAC TTA ATC AGC AAT ATC AAC GTA ATA GTT CTG GAA CTA AAG GGA1681TCC GAA ACA ACA TTC ATG TGT GAA TAT GCT GAT GAG ACA GCA ACC ATT GTA GAA TTT CTG1741

nucleotide 1 to 57 (striked out): leader sequence nucleotide 1387 to1385: IL-2 sequence last nucleotide (striked out): stop codonPeptide Sequence (Incl. Signal Peptide)

The signal peptide is split off during post translational processing andis not part of the final recombinant protein anymore.

Light Chain (SEQ ID NO: 7): 1

21 V V M T Q T P L S L P V T P G E P A S I 41S C R S S Q S L V H R N G N T Y L H W Y 61L Q K P G Q S P K L L I H K V S N R F S 81G V P D R F S G S G S G T D F T L K I S 101R V E A E D L G V Y F C S Q S T H V P P 121L T F G A G T K L E L K R T V A A P S V 141F I F P P S D E Q L K S G T A S V V C L 161L N N F Y P R E A K V Q W K V D N A L Q 181S G N S Q E S V T E Q D S K D S T Y S L 201S S T L T L S K A D Y E K H K V Y A C E 221V T H Q G L S S P V T K S F N R G E C Heavy Chain(incl. IL-2; SEQ ID NO: 8): 1

21 V Q L V Q S G A E V E K P G A S V K I S 41C K A S G S S F T G Y N M N W V R Q N I 61G K S L E W I G A I D P Y Y G G T S Y N 81Q K F K G R A T L T V D K S T S T A Y M 101H L K S L R S E D T A V Y Y C V S G M E 121Y W G Q G T S V T V S S A S T K G P S V 141F P L A P S S K S T S G G T A A L G C L 161V K D Y F P E P V T V S W N S G A L T S 181G V H T F P A V L Q S S G L Y S L S S V 201V T V P S S S L G T Q T Y I C N V N H K 221P S N T K V D K R V E P K S C D K T H T 241C P P C P A P E L L G G P S V F L F P P 261K P K D T L M I S R T P E V T C V V V D 281V S H E D P E V K F N W Y V D G V E V H 301N A K T K P R E E Q Y N S T Y R V V S V 321L T V L H Q D W L N G K E Y K C K V S N 341K A L P A P I E K T I S K A K G Q P R E 361P Q V Y T L P P S R E E M T K N Q V S L 381T C L V K G F Y P S D I A V E W E S N G 401Q P E N N Y K T T P P V L D S D G S F F 421L Y S K L T V D K S R W Q Q G N V F S C 441S V M H E A L H N H Y T Q K S L S L S P 461G K A P T S S S T K K T Q L Q L E H L L 481L D L Q M I L N G I N N Y K N P K L T R 501M L T F K F Y M P K K A T E L K H L Q C 521L E E E L K P L E E V L N L A Q S K N F 541H L R P R D L I S N I N V I V L E L K G 561S E T T F M C E Y A D E T A T I V E F L 581N R W I T F C Q S I I S T L T amino acid 1 to 19 (striked out): leadersequence amino acid 463 to 595: IL-2 sequence

Two GMP compliant batches of the ch14.18/CHO (APN311) antibody have beenproduced. These two batches of the drug that have been produced are LotT651204-A (containing 4.3 ml (4.6 mg/ml) antibody) and Lot T900310-A(containing 4.5 ml (4.5 mg/ml) antibody. The APN311 monoclonal antibodybulk preparation is manufactured as a concentrate for the preparation ofIV infusions.

TABLE 7 Composition of the final APN311 preparation Product Mouse-humanchimeric monoclonal anti-GD2 lgG1 antibody Name (ch 14.18/CHO; APN311)Content 4.25-4.75 mg/ml (the exact content per mL may slightly vary fromlot to lot and is given on each vial) Buffer 20 mM histidine, 5%saccharose, 0.01% Tween 20, WFI pH Value 5.5-6.5 Excipient NonePreparation Guide

The antibody must be prepared under sterile conditions. The appropriatevolume of ch14.18/CHO antibody (APN311) should be withdrawn from thevials. It is recommended that the antibody solution is filtered (0.2 to1.2 m) before injection into the patient either by using an in-linefilter during infusion (as some centres do routinely) or by filteringthe solution with a particle filter (e.g. filter Nr. MF1830,Impromediform, Germany). The volume of the antibody is added to aninfusion bag containing NaCl 0.9% and 1% human serum albumin (HSA), e.g.100 ml NaCl 0.9% and 5 ml human serum albumin 20%.

Calculation of the quantity of ch14.18/CHO (APN311) to be diluted

The amount of ch14.18/CHO (APN311) to be administered is calculated asfollows: Dosage: 10 mg/m²/day, day 8-17, as 24 h infusion.

Example calculation: If a patient has a body surface area (BSA) of 0.7,he/she needs 7 mg (10×0.7) per day, or 70 mg for ten treatment days (onecycle).

Example 2: CDC Assay Method

Principle for CDC (Complement Dependent Cytotoxicity)

Induction of tumor cell cytotoxicity of normal human serum or plasma inthe presence of APN301 or APN311, or of patients' serum or plasma afterinfusion of one of these antibodies, to the GD2 antigen positive LAN-1neuroblastoma cancer cell line (target cells) was determined in a⁵¹Chromium release assay.

The target cells were incubated with Na₂ ⁵¹Cr(VI)O₄, which permeates thecell membrane and binds to cytoplasmatic proteins in the reducedCr-III-valent form, thereby not leaking out anymore of an intact cell.When these cells are lysed after incubation with serum or plasma andantibodies or patients' serum or plasma, radioactivity is released intothe supernatant dependent on the lytic capacity in the tested samples.

Spontaneous background lysis and total lysis (maximally achievable celllysis or maximal possible target cell lysis) by a surfactant weredetermined in each individual experiment. After subtracting spontaneouslysis, the lysis induced by the tested samples was calculated as % oftotal lysis.

Serum or Plasma Sampling:

Whole blood from normal human donors or from patients was sampled usingheparinized vacutainer vials for plasma or serum clotting vials forserum. Vials were centrifuged at 2000 g for 20 minutes. The supernatantplasma or serum could be used immediately for the assay or stored at−20° C. (no thawing and re-freezing allowed).

Labeling of Target Cells with ⁵¹Cr:

LAN-1 cells were cultivated in RPMI 1640 with 10% heat inactivated FCS.The day preceding the assay they were transferred into fresh flasks andfresh medium.

The assay was carried out in a 96-well flat bottom cell culture plate,using 4×10⁴ labeled cells per well with an activity of 800 nCi ⁵¹Cr perwell.

The needed amount of cells was harvested from the culture flasks, thesuspension centrifuged and re-suspended in 1 ml of PBS def. with 0.1%EDTA and 1% FCS. The calculated volume of the ⁵¹Cr solution was added,cells were incubated for 90 minutes at 37° C. and 5% CO₂ under gentlerotation of the tube.

Then the cell suspension was washed twice with cell culture medium toremove radioactivity from outside the cells. This medium containedadditionally 100 U/ml penicillin G and 100 μg/ml streptomycin sulphate.The pellet of labeled cells after the washing steps was resuspended tothe wanted concentration of 4×10⁵ cells per ml.

Assay Procedure:

For the assessment of cytolytic capacity of antibodies, the followingwas pipetted:

-   -   50 μl of the samples (antibody dilutions)    -   100 μl 1:4 pre-diluted normal human serum or plasma    -   100 μl ⁵¹Cr labeled cell suspension (4×10⁵ per ml)

For the assessment of cytolytic capacity of patients' plasma or serum,the following was pipetted:

-   -   50 μl medium    -   100 μl 1:4 pre-diluted patients' plasma or serum    -   100 μl ⁵¹Cr labeled cell suspension (4×10⁵ per mL)

Assay plates for CDC were incubated in a CO₂ incubator at 37° C., 5%CO₂, for 4 hours, or when compared directly to a WBT, for 20 hours.

In addition, an aliquot of each blood sample is preincubated withganglidiomab, a monoclonal mouse anti-ch14.18 antibody (anti-idiotypicantibody), and further processed as described above. It bindsspecifically to the antibody present in the blood sample afteradministration of the antibody to the patient. Thereby, theanti-idiotypic antibody inhibits the ability of the antibody to lyse thetumor target cells in combination with blood cells (WBT) and/or plasmacomponents (CDC). Residual lysis can be defined as a non-antibodymediated effect.

Then the supernatants of each well are harvested using harvesting frameswith absorption cartridges and a harvesting press (skatron). Thesecartridges soaked with the cell supernatants are transferred intocounting vials of the gamma counter. Radioactivity, which isproportional to the release of 51-chromium after a damage of the labeledtarget cells, is measured from all samples and expressed in counts perminute (cpm). Results are calculated as % lysis subtracting the cpms ofspontaneous lysis from all sample values and relating to the cpm of themaximally achievable lysis with a surfactant which is 100%.100×(cpm sample minus cpm spontaneous lysis)=% lysis of samples

cpm maximal lysis minus spontaneous lysis

The above described CDC assay method has been used for the results asshown in FIGS. 1, 2, 3, 5, and FIG. 33 A.

A similar CDC assay method has been used for the results as shown inFIG. 7, however, calcein has been used as label for the LAN-1 cellsinstead of chromium.

Example 3: WBT Method

Principle for WBT (Whole Blood Test):

Induction of tumor cell cytotoxicity of normal human whole blood in thepresence of APN301 or APN311, or of patients' whole blood after infusionof one of these antibodies, to the GD2 antigen positive LAN-1neuroblastoma cancer cell line (target cells) was determined in a⁵¹Chromium release assay.

The target cells were incubated with Na₂ ⁵¹Cr(VI)O₄, which permeates thecell membrane and binds to cytoplasmatic proteins in the reducedCr-III-valent form, thereby not leaking out of intact cells anymore.When these cells are lysed after incubation with whole blood andantibodies or patients' whole blood, radioactivity is released into thesupernatant dependent on the lytic capacity in the tested samples.

Spontaneous background lysis and total lysis (maximally achievable lysisor maximal possible target cell lysis) by a surfactant were determinedin each individual experiment. After subtracting spontaneous lysis, thelysis induced by the tested samples was calculated as % of total lysis.

Blood Sampling:

Whole blood from normal human donors or from patients was sampled usingheparinized vacutainer vials.

Labeling of Target Cells with ⁵¹Cr:

LAN-1 cells were cultivated in RPMI 1640 with 10% heat inactivated FCS.The day preceding the assay they were transferred into fresh flasks andfresh medium.

The assay was carried out in a 96-well flat bottom cell culture plate,using 4×10⁴ labeled cells per well with an activity of 800 nCi ⁵¹Cr perwell.

The needed amount of cells was harvested from the culture flasks, thesuspension centrifuged and re-suspended in 1 ml of PBS def. with 0.1%EDTA and 1% FCS. The calculated volume of the ⁵¹Cr solution was added,cells were incubated for 90 minutes at 37° C. and 5% CO₂ under gentlerotation of the tube.

Then the cell suspension was washed twice with cell culture medium toremove radioactivity from outside the cells. This medium containedadditionally 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate.The pellet of labeled cells after the washing steps was re-suspended tothe wanted concentration of 4×10⁵ cells per ml.

Assay Procedures:

For the assessment of cytolytic capacity of antibodies the following waspipetted:

-   -   50 μl of the samples (antibody dilutions)    -   100 μl 1:2 pre-diluted normal human whole blood    -   100 μl ⁵¹Cr labeled cell suspension (4×10⁵ per ml)

For the assessment of cytolytic capacity of patients' whole blood thefollowing was pipetted:

-   -   50 μl medium    -   100 μl 1:2 pre-diluted patient's blood    -   100 μl ⁵¹Cr labeled cell suspension (4×10⁵ per ml)

Assay plates are incubated in a CO₂ incubator at 37° C., 5% CO₂, for 20hours.

In addition, an aliquot of each blood sample is preincubated withganglidiomab, a monoclonal mouse anti-ch14.18 antibody (anti-idiotypicantibody), and further processed as described above. It bindsspecifically to the antibody present in the blood sample afteradministration of the antibody to the patient. Thereby, theanti-idiotypic antibody inhibits the ability of the antibody to lyse thetumor target cells in combination with blood cells (WBT) and/or plasmacomponents (CDC). Residual lysis can be defined as a non-antibodymediated effect.

Then the supernatants of each well are harvested using harvesting frameswith absorption cartridges and a harvesting press (skatron). Thesecartridges soaked with the cell supernatants are transferred intocounting vials of the gamma counter. Radioactivity which is proportionalto the release of 51-chromium after a damage of the labelled targetcells is measured from all samples and expressed in counts per minute(cpm). Results are calculated as % lysis subtracting the cpms ofspontaneous lysis from all sample values and relating to the cpm of themaximally achievable lysis with a surfactant which is 100%.100×(cpm sample minus cpm spontaneous lysis)=% lysis of samples

cpm maximal lysis minus spontaneous lysis

The above described WBT method has been used for the results as shown inFIGS. 1, 2, 3, 4, and FIG. 33 B.

Example 4: Patient Treatment with a Continuous Intravenous Infusion of aPreparation Comprising an Anti-GD2 Antibody

In a compassionate use setting of the treatment with ch14.18/CHO(APN311) in 41 patients with relapsed or refractory neuroblastoma, acontinuous infusion modality has been used in order to possibly reducethe pain always associated with anti-GD2 antibody immunotherapy, inconjunction with s.c. IL2 and isotretinoin. Clinical responses weredetermined by local physicians based on evaluations ofmetaiodobenzylguanidine scintigraphy (mIBG), magnetic resonance imaging(MRI) or X-ray computed tomography (CT), bone marrow histology (assessedby aspirate or trephine biopsy) and catecholamines.

mIBG: 31 out of 41 patients had disease detected in mIBG beforeimmunotherapy.

5 of these 31 patients (16%) had a complete response (CR), 7 (23%) had apartial response (PR), 4 (13%) had stable disease (SD) and 13 (42%) hadprogressive disease (PD). 2 patients (6%) are not evaluable. For one ofthem no mIBG examinations after immunotherapy were available, the otherpatient had the remaining tumor removed by surgery before restagingassessments after immunotherapy were done.

Overall, in 12 out of 31 patients (39%) with detectable disease in mIBGbefore immunotherapy a response (CR or PR) was detected afterimmunotherapy.

Furthermore, in 3 out of the 13 patients with PD after completion ofimmunotherapy (23%), a PR was detected after the 3rd immunotherapycycle.

MRI/CT: 13 out of 41 patients had detectable disease in MRI or CT insoft tissue before immunotherapy.

One additional patient with a positive MRI at baseline was not evaluableas the remaining tumor was completely resected before final restagingassessments. After 3 immunotherapy cycles this patient had SD.

5 of the 13 patients with detectable disease in MRI or CT beforeimmunotherapy (38%) had a PR, 4 (31%) had SD and 3 (23%) had PD.Evaluation of one patient (8%) is pending.

Bone marrow: 19 out of 41 patients had detectable disease in bone marrowbefore immunotherapy. 4 of these 19 patients (21%) showed a responseafter immunotherapy in the respective tests. Additionally, in 6 patients(32%) a response was detected after 3 immunotherapy cycles. PD, however,was noticed in 4 of these patients in other examinations after 3 cycles,and in 2 of these patients at the end of the immunotherapy.

Catecholamines: 18 out of 41 patients had increased catecholamine levels(Vanillyl mandelic acid (VMA) and/or Homovanillic acid (HVA)) beforeimmunotherapy. In 7 of these 18 patients (39%) normal catecholaminelevels were detected after 3 cycles of immunotherapy and/or aftercompletion of immunotherapy.

In addition to these marked response rates observed in therelapsed/refractory patients treated under a compassionate use settingby the continuous infusion modality, in all of these patients animpressive reduction of the pain side effect was noticed, allowing tosubstantially reduce or even completely avoid treatment with morphine:

The standard dose of i.v. morphine for this schedule is 30 μg/kg/h. Inpatients receiving ch14.18/CHO (APN311) continuous infusions,significantly less i.v. morphine was used compared to patients whoreceive ch14.18/CHO (APN311) bolus infusions. In many patients it waseven possible to discontinue i.v. morphine completely and to treat painwith oral Gabapentin only. The morphine use during ch14.18/CHO (APN311)continuous infusions is displayed in FIG. 10 and Table 9. Antibodyinfusions were always initiated on Day 8. The actual morphine dose (meanof 37 patients) per overall treatment time (comprising all 6 treatmentcycles) was 5.4 mg/kg compared to the prescribed morphine dose of 13.5mg/kg of a previous phase I clinical study (0.9 mg/kg/day, infused over8 h per day for 5 days per cycle, 3 cycles), and compared to 10 mg/kg ofan ongoing phase III clinical study, both with non-continuous antibodyinfusion schedules (0.48 mg/kg/day on the first day and 0.38 mg/kg/dayon the subsequent treatment days, infused over 8 h per day for 5 daysper cycle, 5 cycles).

In addition, a Phase I/II study administering APN311 by continuousinfusion combined with subcutaneous aldesleukin (IL2) in patients withprimary refractory or relapsed neuroblastoma) has been set up to

-   -   reduce the toxicity (pain) profile whilst maintaining        immunomodulatory efficacy of ch14.18/CHO mAb (APN311) treatment        in combination with a fixed dose of s.c. IL2.    -   reduce the toxicity (pain) by establishing a continuous infusion        scheme over 10 to 21 days at up to three dose levels (total        doses: 100 mg/m²-150 mg/m²-200 mg/m²).    -   Improve patient compliance.    -   Keep or even improve efficacy of immune therapy.

Preliminary results from this Phase I/II study show that the use ofopioids, especially morphine, to control the massive incapacitating painfrequently occurring during the treatment with GD2 specific antibodies(including APN311) is significantly lower in these patients alreadyduring the first infusion cycles. From the 3^(rd) cycle onwards it maybe even possible to completely refrain from the standard morphineadministration since patients will not require it due to the increasedtolerability of the medication due to the improved application scheme.

The significantly reduced doses of morphine cause less of the opioidtreatment related adverse effects and therefore even allow anout-patient treatment setting, which in turn will positively influencethe ability of the pediatric patients to follow the normal lifestyle ofchildren, e.g. ability to play and attend school, etc.

TABLE 8 Blood samples analysed with a WBT shown in FIGS. 11 to 16. dayof treatment patient (within the treatment cycle) cycle MJ 15 I 8 II 10II NG before — 8 I 10 I 15 I JK 8 III 10 III 15 III MM 15 III GA 8 V 10V CJJ 10 V

Blood samples taken at the beginning (i.e. on the first day) of thetreatment period with APN311 (corresponding to day 8 of the treatmentcycle) were taken prior to the start of the APN311 treatment.

TABLE 9 Morphine administration dose per cycle in Cycle Day 8 Day 9 Day10 Day 11 Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 mg/kg % of standardinfus. rate 1 100%  96% 84% 65% 41%  14%  5% 3% 2% 1% infusion rate inmcg/kg/h 30.04  28.93  25.19  19.56    12.26   4.10   1.60   1.00   0.60  0.29 daily dose in mg/kg 0.72 0.69 0.60 0.47   0.29   0.10   0.04  0.02   0.01   0.01 2.97 % of standard infus. rate 2 72% 59% 38% 19% 9%3% 0% 0% 0% 0% infusion rate in mcg/kg/h 21.61 17.13 9.53 3.73   1.06  0.13   0.01 0 0 0 daily dose in mg/kg 0.52 0.41 0.23 0.09   0.03  0.00   0.00 0 0 0 1.28 % of standard infus. rate 3 30% 28% 18% 10% 3%1% 0% 0% 0% 0% infusion rate in mcg/kg/h 9.01 8.10 4.53 1.96   0.37  0.04 0 0 0 0 daily dose in mg/kg 0.22 0.19 0.11 0.05   0.01   0.00 0 00 0 0.58 % of standard infus. rate 4 22% 15%  8%  2% 0% 0% 0% 0% 0% 0%infusion rate in mcg/kg/h 6.61 4.34 2.02 0.39 0 0 0 0 0 0 daily dose inmg/kg 0.16 0.10 0.05 0.01 0 0 0 0 0 0 0.32 % of standard infus. rate 518% 14%  8%  4% 0% 0% 0% 0% 0% 0% infusion rate in mcg/kg/h 5.41 4.052.02 0.78 0 0 0 0 0 0 daily dose in mg/kg 0.13 0.10 0.05 0.02 0 0 0 0 00 0.29 % of standard infus. rate 6  0%  0%  0%  0% 0% 0% 0% 0% 0% 0%infusion rate in mcg/kg/h 0   0   0   0   0 0 0 0 0 0 daily dose inmg/kg 0   0   0   0   0 0 0 0 0 0 0.00

Example 5: Patients Treated with APN311 without IL-2 (and without OtherCytokines) in Comparison to Data of Yu et al. 2010, NEJM (Cited Above)

The data underlying this example have been generated in the High RiskNeuroblastoma Study 1.5 of SIOP-Europe (SIOPEN), EudraCT number2006-001489-17. Patients with high risk neuroblastoma aftermyeloablative therapy (MAT) and autologous stem cell rescue (ASCR), andas the case may be, also other treatments, such as radiation, otherchemotherapies, surgery etc., have received up to 5 cycles of 20mg/m²/day ch14.18/CHO administered as an 8 hour i.v. infusion for 5consecutive days (days 22 to 26 of the treatment cycle) in 28-daycycles. This corresponds to a total dose of 100 mg/m²/cycle ch14.18/CHO.cis-RA has been administered orally twice a day in a dose of 160mg/m²/day on days 1 to 14 of the treatment cycle (in two equal doses perday, i.e. 2×80 mg/m²). In one group, no IL-2 has been administeredduring a treatment cycle and during the overall treatment time. Thepatients of the other group received IL-2 at a dose of 6×10⁶ IU/m²/dayon days 15 to 19 and 22 to 26 of a treatment cycle. The patients weretreated in 6 treatment cycles, wherein treatment cycles 1 to 5 were asdescribed above, and the sixth treatment cycle comprised the treatmentperiod with isotretinoin only (no antibody and no IL-2 administered).

Event-free and overall survival was analyzed. EFS and OS were alsoanalyzed in the subgroup of patients who presented as completeresponders at the start of antibody treatment (no evidence of tumor byany applied examination). The EFS and OS data of this subgroup has beencompared to the EFS and OS data of a treatment schedule with antibody,IL-2 and GM-CSF, and isotretinoin (cis-RA), as described in Yu et al.(NEJM 2010, cited above), and are included in Table 10.

The patients in Yu et al. have been treated with ch14.18/Sp2/0 given ina dose of 25 mg/m²/day on four consecutive days, each dose ofch14.18/Sp2/0 is infused i.v. over 5.75 hours or 10 h (may have beenextended to up to 20 h for anticipated toxicities). IL-2 is given everysecond cycle (e.g. cycle 2 and 4) as a continuous i.v. infusion over 4days (96 h) in 3 MIU/m²/day in week 1 of the treatment cycle and in 4.5MIU/m²/day in week 2 of the treatment cycle (i.e. 30 MIU/m²/cycle).GM-CSF is given s.c. in a daily dose of 250 mcg/m² for 14 days (from Day0 through 13). The overall treatment time comprises 5 treatment cycles.See also Tables 2, 3, and 4 for the respective treatment schedules. TheEFS and OS Figures of Yu et al. that are depicted here in FIGS. 19 and20 for comparison include only the complete responders of the abovedescribed treatment.

Therefore, only the subgroup of complete responders to the inventivetreatment have been used for direct comparison.

TABLE 10 2-year event-free survival (EFS) and overall survival (OS) ofcomplete responders (CR) of APN311-302 in comparison to Yu et al. (NEJM2010, cited above) in percent 2 y 2 y EFS in % OS in % Subgroup of CR atstart of randomization in 70 81 APN311-302 study: APN311 + cis-RA NEJM2010: ch14.18 + IL2 + GM-CSF + cis-RA 66 86 NEJM 2010: cis RA only 46 75

The results are also depicted in FIGS. 18, 19, and 20 (also incomparison to Yu et al.) and in FIGS. 21 to 32 (for further detailsabout toxicities, i.e. adverse effects or side effects observed).According to these data, IL-2 does not contribute to the efficacy ofanti-GD2 antibody treatment. However, concomitant IL-2 treatmentsubstantially increases side effects and toxicities. APN311+cis-RAtreatment of complete responders at start of immunotherapy leads tosimilar 2-year event-free and overall survival percentages and totalsurvival curves than those found in the ch14.18/SP2-0+IL2+GM-CSF+cis-RAgroup of the NEJM 2010 study (Yu et al. 2010, cited above).

This suggests that the addition of both IL2 and GM-CSF are not needed toachieve the efficacy level of the combination treatment as described inNEJM 2010.

Both EFS and OS for APN311+cis-RA (complete responders at start ofimmunotherapy) compared to EFS and OS of patients of NEJM 2010 whoreceived cis-RA only are improved (2y EFS: 70% versus 46%; 2y OS: 81%versus 75%; see also table 7). This indicates single agent efficacy ofAPN311.

In an overall assessment of risk and benefit, it is therefore concludedthat APN311+cis-RA in complete responders at start of immunotherapy maybe even superior to the 4-component immunotherapy as described in NEJM2010, especially because of the substantially reduced side effectprofile and reduced complexity of treatment due to omission of IL2 andGM-CSF, and because of the similar clinical efficacy in terms of EFS andOS.

Example 6: CDC and WBT Assay of a Neuroblastoma Patient Treated withAntibody

A neuroblastoma patient has been treated with APN311 in a continuousinfusion schedule, i.e. has received the antibody as a continuousinfusion over 24 h per day for 10 consecutive days (on days 1-10 of atreatment cycle) in a dose of 10 mg/m²/day for 6 subsequent cycles. Eachtreatment cycle comprised 35 days. During each treatment cycle andduring the overall treatment time of said 6 subsequent cycles, thepatient has only received antibody treatment and has not been treatedwith any cytokine nor retinoid.

Blood samples have been obtained from said patient at the indicated timepoints and have been analyzed in a CDC assay and WBT as described inExamples 2 and 3. The results are depicted in FIG. 33. FIG. 33 showsthat antibody treatment without any cytokine treatment causes increasedlevels of tumor cell lysis. These levels of tumor cell lysis arecomparable to the levels of patients treated with the antibody and IL-2(one representative patient is shown in FIG. 34). The patient depictedin FIG. 34 has been treated with the same treatment schedule as thepatient shown in FIG. 33, but has additionally been treated with s.c.IL-2 in a dose of 6×10⁶ IU/m²/day on days 1 to 5 and 8 to 12.

As can be seen especially in FIG. 33 B, an increased level of targetcell lysis in the whole blood test (WBT) is maintained even for timeperiods within the overall treatment time, where the patient is nottreated with the preparation comprising an anti-GD2 antibody, i.e. inthe intervals between the treatment periods with the preparationcomprising an anti-GD2 antibody.

The invention claimed is:
 1. A method of treating neuroblastoma in ahuman patient comprising administering ch14.18 antibody in at least onetreatment cycle, the at least one treatment cycle comprising at leastone treatment period and optionally one or more days of noadministration of the ch14.18 antibody to the patient between repeatedtreatment periods, wherein the treatment period is one or moreconsecutive days when the ch14.18 antibody is administered to thepatient, the ch14.18 antibody being administered in a dose of or between10 to 20 mg/m²/day by intravenous infusion over 10 to 24 hours per dayto the patient without administering IL-2 within the same one or moretreatment cycles, wherein the ch14.18 antibody comprises a heavy chainand a light chain, the heavy chain having an amino acid sequencecomprising SEQ ID NO:4 and the light chain having an amino acid sequencecomprising SEQ ID NO:3, and wherein a neuroblastoma is treated in thepatient.
 2. The method of claim 1, further comprising treating thepatient one or more times with a retinoid preceding, accompanying,and/or following administering of the ch14.18 antibody.
 3. The method ofclaim 1, wherein the ch14.18 antibody is administered to the patient asa continuous intravenous infusion over 24 hours per day for one or moredays.
 4. The method of claim 1, wherein the ch14.18 antibody isadministered to the patient for two or more treatment cycles.
 5. Themethod of claim 1, wherein the patient is not treated with GM-CSF withinthe same one or more treatment cycles.
 6. The method of claim 1, whereinthe patient is not treated with a cytokine within the same one or moretreatment cycles.
 7. The method of claim 1, wherein the patient is nottreated with IL-2, and/or GM-CSF, and/or a cytokine within the same oneor more treatment cycles and/or within an overall treatment time thatcomprises one or more treatment cycles and optionally one or more daysof no administration of the ch14.18 antibody to the patient betweenrepeated treatment cycles.
 8. The method of claim 1, wherein theneuroblastoma is high risk neuroblastoma, neuroblastoma stage 4, minimalresidual neuroblastoma disease, relapsed neuroblastoma, and/orrefractory neuroblastoma.
 9. The method of claim 1, wherein the ch14.18antibody administered to the patient is a CHO cell or SP2/0 cellproduced antibody.
 10. The method of claim 1, wherein the ch14.18antibody is administered in a daily dose of 10 or 20 mg/m².
 11. Themethod of claim 1, wherein the ch14.18 antibody is administered for 4,10, 14, 15, or 21 consecutive days.
 12. The method of claim 1, whereinthe ch14.18 antibody is administered in a preparation further comprisingsucrose, polysorbate 20, histidine, and hydrochloric acid.
 13. Themethod of claim 12, wherein the preparation comprises 4.5 mg/mL ch14.18antibody, 50 mg/mL sucrose, 0.1 mg/mL polysorbate 20, and 3.1 mg/mLhistidine.
 14. The method of claim 1, wherein the ch14.18 antibody isadministered in a dose of 10 mg/m²/day for 10 consecutive days for 1, 2,3, 4, 5, 6 or more treatment cycles.
 15. The method of claim 1, whereinthe ch14.18 antibody administered to the patient is a CHO cell producedch14.18 antibody.
 16. The method of claim 1, wherein the ch14.18antibody administered to the patient is a SP2/0 cell produced ch14.18antibody.
 17. The method of claim 1, wherein the administration of thech14.18 antibody is accompanied by the administration of a reduced doseof morphine and/or one or more other analgesic.
 18. The method of claim1, wherein the ch14.18 antibody is administered in a dose of 60, 70, 75,80, or 100 mg/m²/treatment cycle.
 19. The method of claim 1, wherein thepatient is not treated with IL-2 within an overall treatment time thatcomprises one or more treatment cycles and optionally one or more daysof no administration of the ch14.18 antibody to the patient betweenrepeated treatment cycles.
 20. The method of claim 1, wherein thech14.18 antibody is a humanized ch14.18 antibody.
 21. The method ofclaim 1, wherein the patient is not treated with IL-2, GM-CSF, or acytokine within an overall treatment time that comprises one or moretreatment cycles and optionally one or more days of no administration ofthe ch14.18 antibody to the patient between repeated treatment cycles.